JPH04500122A - Polypeptide-triggered monoclonal receptors for protein ligands - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] For applications related to polypeptide-induced monoclonal receptors for protein ligands cross reference This application was filed on August 12, 1988 and is pending application serial No. 232,395, which was filed on November 9, 1987. Pending application serial number.

118.823, which was filed on April 16, 1987. This is a partial continuation application with serial number 039,534 filed under Pending application serial number 736,54 filed on May 21, 1985 5, which is a continuation in part of the application filed on August 17, 1984. Partly pending PCT application PCT/US84101304, its U.S. national phase filed on February 15, 1985, with serial number 713.410, the application is Pending U.S. Application Serial N, filed August 17, 1983 and abandoned. o, 524.084, a continuation-in-part application.

Interest in cancer genes has increased over the past decade. RNA tumor virus has been associated for more than 50 years as a causative agent for experimentally inducing neoplasia in chickens. However, the mechanism of virus-induced neoplasia began to appear on the 19th. It wasn't until the mid-'70s [Bishop (1983) Ann, Rev., Biochem; 52.301-54]. According to one such mechanism, replication-competent avian viruses and 4JII mammalian viruses lacking cellular genes that confer transforming potential to the virus. Acquire a gene.

Technical field The present invention relates to immunological receptors and ligands, and more particularly to their amino acid residues. For a polypeptide whose base sequence corresponds to that of a retroviral oncoprotein ligand. Concerning monoclonal receptors generated by

Background technology Retroviruses are viruses that contain a single strand of RNA rather than DNA as their genomic material. It is The single-stranded RNA genome of each of these viruses is After infecting the host, double-stranded DN A is generated. This DNA of the virus genome The replica then permanently inserts itself into the chromosomes of the successfully infected cell and this Replicates in the host chromosome.

The retroviruses described below and in the claims furthermore have replication defective It can be defined as a torovirus. In other words, these viruses The RNA genome of the virus is introduced into the chromosome of the infected host and translated into DNA. It does not contain its own gene encoding the reverse transcriptase enzyme normally required to enable stomach. Additionally, the retroviruses described below are typically replication-competent during their infection. It must be assisted by a powerful so-called helper virus. This second viruses successfully transfer genomic material from both viruses into infected host cells. It contains a gene encoding a reverse transcriptase that is integrated to transform the cell.

For ease of understanding, hereinafter and in the claims, replication defective retroviruses are referred to as Although described simply as retroviruses, these are replication-defective and are not infectious. requires the help of helper viruses for success and transformation of host cells. I hope you understand what I mean. This usage of the term retrovirus is well known in the art. has been used in the art without further explanation.

Some retroviruses can cause the formation of solid tumors within a short period of time after being inoculated into the host. It is highly carcinogenic as judged by its ability to cause cancer.

These viruses also cause "cancerous" changes in cells grown and cultured in the laboratory. can be caused. Such changes are called “transformation,” and the oncogenic virus provides a reliable in vitro applied biological evaluation of Viruses like this Several species have been isolated from chickens, turkeys, mice, rats, cats and monkeys.

A single gene, an oncogene, on the genome of these highly oncogenic viruses , which is responsible for the tumor-causing ability of the virus. For some viruses, this The protein products of these oncogenes (hereinafter referred to as oncoproteins or oncobrotiins) are Serum from animals with virus-induced tumors has been shown to contain antibodies against these oncoproteins. It has been immunologically identified using this fact.

A rapidly growing body of evidence suggests that retroviral oncogenes control all vertebrate Closely related to and derived from a specific gene site in normal cell genetic information Which indicates that.

Similarities between the gene product of the sis oncogene and the platelet-derived growth factor chain [Dourit et al. Le et al. (1983) Science 221.275-277°Waterfield (1983) Nature 304.35-39], malignant traits caused by oncogenes. Provides the strongest link between conversion and the promotion of normal cell division by growth factors . The identity between this oncogene product, growth factors, and cellular receptors suggests that erbB This was further corroborated by sequence analysis of the epidermal growth factor cell receptor, which was found to be a common homologue. [Downward et al. (1984) Nature 307.521-527, Ulli (1984) Nature 309.418-425]. Furthermore, fms anti body and colony-promoting factor-1 receptor [Schiller et al. (1985) Cell, 665-6 76, as well as protein kinase similarity and insulin receptor [Ullrich et al. 985) Nature 313.756-761 and many sequenced oncobrothi The imaginal factor receptor showed the kinase activity of Yin [Yardon et al. (1986) Nature -323,226-232], the immunological cross-reactivity of the signals of several adult factors may be important for transduction.

acquired by retroviruses or identified by performing transfection. Sequencing of cancer genes that have been studied has expanded (exceeded) the number of kinase families [Hunter et al. (1985) Ann, Shiv. Biochem, 54.897-930] This sequence analysis There are a large number of kinase-related proteins, and the number of families is determined based on sequence identity and overall structural similarity. It was suggested that they could be divided into several subgroups. The kinase family is conveniently with or without extracellular (hormones/growth/factors) binding main It is divided into gene products.

The great similarity between the src and yes kinase proteins has emerged over the last few years. There is. [Kitamura et al. (1982) Nature 297.205-208 Recently, Sea-fencing of additional genes reveals this identity in fgrs [S-7 Hao et al., ( 1984) Science 222.63-66] Ick, [Manni et al., (198 6) Brok, Natl, Akado, Sai, Ninisni 83.5459-5463 and iyn [Yamanashi et al. (1987) Mol, & Sell Paiol, L. The number has expanded to 237-243. All six genes have approximately the same size 5 5-65kd proteins are coded, and the genes are encoded as if they were the same ancestral prototype. Divided into intron/exon regions indicating that they evolved from oncogenes . However, each gene is located on a separate chromoseme and in different tissues. express different proteins.

Many additional kinase families can also be placed into subgroups.

was [Pan-Beveran et al. (1981) Nature 298.258-262 ] is deep in pin-1 [Selten et al. (1986) cells 46.603-611]. Related, it is a preferred integration site for Moloney leukemia virus. aM[Rep. Di et al. (1983) Brok, Natl, Aceto.

Sai, Ninisny 80.3623-3627 are revised [Crew et al. (1986 ) is closely related to Science 234.1545-1547. fes [ham Bu et al. (1982) cells 30.775-785 and fps [Nbuya et al. (198 2) Cells 30,787-795] are mammalian and chicken fragments of the same gene. It is. Similarly, raf [Strive et al. (1984) Nature 309.85 ~88 and miA [Mark et al. (1984) Science 224.285-28 9] are identical genetic mammalian and chicken analogs. These are A- rof/pks [Waleiher et al. (1986) Mol, & Cell Paiol, 6 ．． 2655-2662, Mark et al. (1986) Blok, Natl. Akado, Sai. 83.6312-6316].

A subgroup that does not have a viral component is the protein kinase C1 phorbol es Contains the gene that encodes the receptor for tell. This subgroup is less Both have three closely related genes. [Kousens et al. (1986) Science 233.859-866, Knoff et al. (1986) Cell 46.491-502.

Furthermore, one of the genes encodes two proteins by using alternative exons. [Oates et al. (1987) Nature 325.161-161]. than others A slightly related cytoplasmic kinase is the cAMP- and cGMP-dependent protein kinase [Shoji et al. (1981) Brok, Natl, Akad, Sai, Ninisny 7 8.848-851. Takao et al. (1984) Biochemistry 23.4207 ~4218], similarly, myosin light chain kinase [Takao et al. (1985) Biochem. Mistry 24.6028-6037]. some transmembrane Nases have also been sequenced in the past few years.

Genes closely related to the human epidermal growth factor receptor (HER) are also found in humans (HER -2) [Kousens et al. (1985) Science 230.1132-1139 and rat (neu) [Barbman et al. (1986) Nature 319.226 ~230] were found. ros [Neckermeyer et al. (1985) J., V. 53.879-884], whose sequence is incinilin receptor (HIR) [Ullrich et al. (1985) Nature 313.756-7 Although it is most closely related to 61, it is not well known. colony promotion factor 1 receptor, FMS [Hamp et al. (1984) Bloch, Natl, Akad, Sai; Nini 81.85-89 contains kit and platelet-induced growth factor, PDG. Receptor for E-R [Yardon et al. (1986) Nature 323, 226-232] and form subgroups. In addition, trk [7- Chinzanka et al. (1986) Nature 319.743-748 and met-8 [Digi et al. (198 5) Nature 318.385] The sequence of an oncogene has no known corresponding growth factor. It was announced even though it wasn't.

Similar, but less widespread, advances have been made in binding proteins expressed by the ras oncogene family. It is also found in nucleotides that contain Sequence data were obtained from baS [Reddy et al. (1985 ) J.Virol, 53.984-987] is H-ras [Da et al. (1982 )Science 217.934-937], and if H- [Tsuchida (1982) Science 217.937-939]. After going through a two-way exhaustion -ras can write out two proteins [4A and 4B] into a code [matrix] Tuffglas et al. (1,983) Nature 301.501-506.

The third member, N-ras, is also linked to H- and K-ras in this site. [Taparowski et al. (1983) Cell 34.581-586]. other A closely related gene is R-ras [Rowe et al. (1987) Cell 48.137 ~146], this gene is derived from three ras genes, which are derived from the same ancestral gene. Although closely related, R-ras has different intron/extron Has a son border. Other genes, rho7 [7 Zuru et al. (1985) Cell 41 ．． 31 to 40], sites similar to ras are dispersed. Furthermore, a third group, ral also has a similar site [Chardin et al. (1986) I, 5.2203-2208. Moreover, the yeast gene ypt [Gallwicz et al. 1983) Nature 306.704-707] has a similar site to ras. , this gene is clearly different from two yeast genes that are very similar to ras. Immediately In other words, they are more similar to R-RAS.

Other genes, also similar to ras, are associated with G proteins [Ito et al. (1986) 83.3776-3780F as well. Lansda Kunyong and elongation factor, Tu [Lochley et al. (1985) Science 2 28. Contains 96-99 pieces. G protein promotes (Gs) and Gi) adenylate Composed of subunits that inhibit cyclase. Other related proteins (Go) are Has unknown functions. These proteins are a variety of different proteins with closely related sequences. It exists in the form of

Nuclear protein myb [Rusch et al. (1982) Science 216.1421-14 23, ll1yc [Colby et al. (1983) Nature 301.722-72 5] and fos [Van Straten et al. (1983) Blok, Natl, A. Kado, Rhinoceros, Ninisni 80.3183-3187 Ko are cell positions rather than sequences. and more related to oncogenes from other families. However, these cancer genes Additional genes related to the gene were identified. N-myc [Stanton (1986) Brok, Natl, Akado, Sai, Ninisni 83.1772-1776 and Bire-JIC [Nara et al. (1985) Nature 318.69-73 sequences are Relevant sequences represented and published were identified. Furthermore, the array is f.

Slightly related to S. Related fos (r-fos) [Cochran et al. (1984) 226.1080-1082 sequences have been published, and unpublished data are as follows: Phosphorylase inhibitors limit similarity as the jun oncogene does. and

Other groups of nuclear cancer gene-associated proteins include steroid and thyroid hormone receptors including. The only sequence related to erbA was published [Sabu et al. (1986) Ichar 324.635-640. Weinberger et al. (1986) Nature 3 24.641-646] However, hybridization studies finally revealed two related sequence is present in the human genome [Weinberger et al. (1986) Jah 324.641-646. The steroid receptor sequence is erbA (thyroid protein). lumon receptor) and several receptors (estrogen, gumcoerceide, progesterone). Indicates that it is a part of the jade material containing sterone, aldesterone) [green etc. 1986) Science 231.1150-1153, Hornbarb et al. Nature 318.635-641 and Conley et al. (1986) Science 233.76. 7-770 pieces.

In the growth factor group, only PDGF-1fiJl [Dortle et al. 983) Science 221.275-277 and Waterfield et al. (1 983) Nature 304.35-39] has a similar arrangement to 5is (PDGF-2). Has a column. However, other growth factors [Gregory (1975) Nature 257.325-327, Magaz et al. (1983) Blok, Natl, Akad, Rhinoceros, Nini 80,4684-4688 (EGF and TGF) are e rb binds to the product of the B proto-oncogene, and C3F-1 [Kawasaki et al. (19 85) Science 230.291-296] is linked to the fms proto-oncogene. match. Furthermore, TGF [Prink et al. (1985) Nature 316.701~ 705] is a Mueller inhibitor [Kate et al. (1986) Cell 45.685-6 98] and found in other subgroups and various forms of inhibition for similar reasons. [Meylan et al. (1985) Nature 318.659-663 and Vue (1986) Nature 321.776-779].

Finally, sequences expressing two related associative sites of MMT~7 were published [V Ann Othan et al. (1984) cells 39.233-240 and Moore et al. 86) Embo Shei 5.919-924].

In the past few years, the number of related publications has increased dramatically. these arrays However, there is a limited number of passageways that control cell division and differentiation. This suggests that many different factors are likely involved in this control.

An example of transduction of a small portion of a cellular gene by a retrovirus is er b It is a B oncogene. The erbB oncogene is an ECG receptor, as previously described. Highly similar to parts of the human body [Ullrich et al. Nature 309.418 (198 4)]. Sequence analysis of the entire receptor gene includes the entire intracellular domain, transmembrane The land domain and extracellular domain parts are demonstrated.

Molecular hybridization studies using specific nucleic acid detection means, and subsequent studies (viral cancer genes) Gene cloning by genetic recombination technology of genes and their cellular related materials is a Rovirus oncogene (v-onc) and cell cancers found in all normal vertebrate cells revealed a close relationship between the genes (c-onc).

Molecular analysis of some of these well-isolated retroviruses has shown that more than one virus revealed different cancer genes. In many cases, the corresponding cellular retrovirus The rus oncogene or oncobrotiin was isolated. For example human EJ or T 24 Bladder cancer genes are associated with the Barbey mouse sarcoma virus (RAS). is a homolog of the transforming genes of Ma) and BALB sarcoma virus (bas). (Parada et al., Nature, 297.474-478 (1982); Dell et al., Brok Natl Acad Sai Ninisny, 79.3627-36 34 (1982); and Santos et al., Nature, 298.343-347 (1 982)). In addition, the oncogene of the human carcinoma cell line LX-1 is associated with the mouse sarcoma virus. Homologous to the transforming gene of the Kirsten strain of rasKi (Dell et al., supra). In addition, it is called fps of avian origin. The same V-OnC as opposed to C-OnC was isolated from a limited number of avian retroviruses. of the object, expressed at least twice: in its mammal, called fes in the cat species. Mammalian homologs have been found in two different strains of feline sarcoma virus.

protein encoded by the viral oncogene and the corresponding homologous protein in the host cell. Both of these proteins are referred to below as oncoproteins, but cellular oncoproteins are typically positive. It is present in small amounts in normal cells, ie it does not need to be associated only with neoplastic conditions. Also, Seki Oncoproteins encoded by related oncogenes can have different molecular weights, so For example, p85 and p encoded by v-fesST and v-fesGA, respectively. 108 oncoproteins and encoded by the c-fes gene in normal mink cells It is thought to be a 100-105 Dalton protein [Sen et al., Blok, Natl. , Akad, Sai, Ninisny, 80.1246-1250 (1983)]. cancer The term protein as used herein refers to proteins whose genes and amino acid residue sequences are at least partially Commonly used for proteins that are partially homologous.

Oncoproteins are generally not present in virus particles that infect cells; It is minimally expressed at best in cells, and more abundantly in neoplastic cells. i.e. cancer Proteins typically are not obtained from viruses. In addition, the separation of cancer proteins from cells is low abundance, complex mixture of proteins found in normal cells and transformed cells. This is difficult because of the relatively small amounts of such proteins present even in isolated cells.

The oncogenes encoded by the v-onc and C-One genes are homologous, but , which nevertheless usually contain large sequences of non-identical amino acid residues. Also, each Oncoproteins encoded by genes of different viral strains that contain the same oncogene In addition, four of the ras genes differing above and at the position of the 12th amino acid residue. in its amino acid residue sequence, as exemplified by the sequence described in the publication. It was found that there was a slight change in In other words, when the cancer protein is in hand. Well, it would be difficult to distinguish between them.

Monoclonal and polyclonal antibodies or idiotype-containing proteins of these antibodies Immunologically induced receptor molecules such as It can be used as a diagnostic agent to evaluate the presence and amount of protein ligands when purifying proteins. and for distinguishing between homologous protein ligands.

The difficulty in obtaining large amounts of oncoproteins is due to the difficulty in obtaining large amounts of cancer proteins encoded by v-fes and v-fps. Cell-induced monoclonal antibodies against whole proteins have been reported by Veronese et al. (J. Bailol, 43.896-904 (1982)), but these oncoproteins This generally precludes the preparation of receptors for Also, the whole protein is Even if it can be used as an immunogen to induce the body's production, large proteins are Polyclonal molecules can be used to target multiple epitopes on large protein branches. Antiserum containing antibodies is produced.

Hybridomas and monochromes that use whole proteins or large protein fragments as immunogens Nuclear antibody technology is useful for narrowing the immunological response to such immunogens. Met. However, such conventional techniques are extremely time-consuming and require Only a relatively small number of hybridomas secrete useful antibodies that recognize it. Also , even if successful, such techniques do not allow the receptor molecule to be raised against it. cannot predict the chemical identity of epitopes. thus recognizing the immunogen specific chemically identified epitopes of protein ligands even after the receptor has been created Obtaining receptors for the moiety ultimately leads to the creation of useful hybridomas. It is a haphazard operation that further reduces the number of

contains 56 amino acid residues and in the amino acid residue sequence for a polypeptide corresponding to the carboxine-terminal portion of the complete interferon molecule. reported the production of monoclonal antibodies (Nature, 294.278 ~280 (1981)). This 56-mer polypeptide has a sequence of approximately compared to one-third.

Arnheiter et al. reported the production of 11 monoclonal antibodies. But this Only one of the 11 monoclonal antibodies of Both bound to all interferon molecules. This binding also 3000-fold excess of intact interferon required to compete with antibodies from TiD Judging by this, it was not very strong. None of the other monoclonal antibodies It did not bind to all molecules.

In addition, the production of hybridomas secreting these monoclonal antibodies was carried out using three immune Requires human liver from infected mice. Desired interferon-binding monochrome The low yields of local antibodies and the fact that these This indicates that the project was relatively unsuccessful.

Lerner et al. used short to intermediate length synthetic amino acid residue sequences as immunogens. successfully obtained protection for animals by the use of vaccines against pathogens using Ta. See Sutcliffe et al., Science, 219.495-497 (1983). I want to be illuminated.

However, until the present invention, hybridomas and their secreted monoclonal receptors The success of preparing a vaccine containing oligoclonal receptors is independent of the success of preparing a vaccine containing oligoclonal receptors. You have to understand something.

That is, for high-yield monoclonal antibody preparation, large amounts of avid ) It is necessary to stimulate B cells to secrete antibodies. On the other hand, synthetic vaccines The broader spectrum of oligoclonal antibodies Something with good affinity will be created. Also, animal protection against pathogens is typical T cells and Both require B cell activation.

Synthetic polypeptides in the production of antibodies that recognize intact proteins and protect animal hosts. A popular explanation for the success of Tide-containing vaccines is that the diversity of immune responses is a rare event. That is, a polypeptide conforms to the conformation of its corresponding sequence in the native molecule. It includes a probabilistic model that allows the observation of the adoption of medium length The concept that polypeptides can often conform to native structures has been Contrary to research. Rather, such polypeptides contain a large number of transitions in dynamic equilibrium. It is thought that it exists as a total of conformational states (an sample). A of this configuration T cell activation by some of the sample samples and B of antibodies raised against some of the samples. Cell production has been thought to be sufficient to confer protection through vaccination .

Summary of the invention The present invention provides (a) a protein ligand encoded by a retroviral gene, and (b) Some amino acid residue sequences of proteins encoded by retrovirus genes. from about 7 to about 40 residues, preferably about 10, with an amino acid residue sequence corresponding to the column. A monochrome that binds to both moderate-length polypeptides of ~30 amino acid residues. nal receptor molecules are considered. This receptor molecule is an immune system that contains polypeptides. arises from (is triggered by) the origin. Most preferably, the receptor molecule is monoclonal receptors such as the IgG or 1gM class of immunoglobulins .

Certain preferred monoclonal receptor molecules of the invention are directed to the genes listed below. Polypeptides listed for proteins encoded by and located in these oncogenes. Combines with putide.

5PYPNLSNQQTR; IGRGNFGEVFSG.

LMEQ (JAYEPGQRPSF: and RKIEIVRKKKPIFKKATV ：　FIVTIFImVRRPPKGKFIRKC:PDGF-I　5IE EAVPAECKTR.

EGF　CLHDGVCMYIELDKYAC;abl　LMRACWQWN PSDRPSF: LGGGQYGEVYEG: and LIFEIATYGMSPYPGIDLSQVY:fms FMQACWALF PTRRPTF: and LGTGAFGLVVEA SRCLMCQCWRKDPEERPTF; LGQGCFGEIMG; and CG55KSKPKDPSQRRRS.

yes LMKLCWKKDPDERPTC; and L position VTKGRVPYPGM VNREvL.

fqr L position TTKGRVPYPGMGNGEVL;bas KLVVVGAK GVGK: 1nt-I LHNNEAGRTTvFS: mil/raf L VADCLKKVREERPLF; and IGSGSFGTVYRG.

ros　LGSGAFGEVYEG　;VWETLTLGQQPYPGLSNI EVL, and Li1TRCWAQDPHNRPTF.

The present invention also provides methods for making monoclonal receptor molecules for protein molecule ligands. Regarding. In this method, preferably a medium length of about 7 to about 40 residues). is a synthetically produced immunogenic polypeptide or this polypeptide conjugated to a carrier. A zygote of the decoder is provided. The amino acid residue sequence of this polypeptide is Corresponds to part of the amino acid residue sequence of This immunogenic polypeptide is Le Limpet is conjugated to a hemocyanin carrier to immunize mice. When used for immunization, a dilution of at least about 1:400 after the third immunization. 50 percent binding titer of immunized mouse serum against polypeptide (b sufficiently immunogenic and antigenic to give a binding titer) . wherein each of the three immunizations contains at least 10 micrograms of the immunization in the conjugate. complete Freund's adjuvant for the first immunization; The second and third immunizations use alum as an adjuvant.

Mammals can use immunogenic polypeptides or conjugates of this polypeptide to carriers. The body is hyperimmunized to a polypeptide at a dilution of at least about 1.400 and Give hyperimmune serum showing a one-cent binding titer. This serum receptor molecule also A polypeptide binds to a protein molecule ligand that corresponds in amino acid base sequence. match.

Hyperimmunized mammals should be treated with a 50 percent dilution of at least about 1:400. After immunization to give a combined titer, a small amount (maintained for about 30 days; , the animal is given a potency-enhancing immunization, such as by intravenous injection.

Antibody-producing cells such as mammalian human liver cells (sprephocytes) with enhanced efficacy , within about 3 to about 5 days from the date of potency-promoting administration to generate hybridoma cells. Fused with myeloma cells. Hybridoma cells created in this way contain protein molecules. Ligand (a part of which the immunogen polypeptide corresponds in amino acid residue sequence) ) is evaluated for the production of monoclonal receptor molecules that bind to Preferably Hybridoma cells also contain monoclonal receptor molecules that bind to polypeptides. is evaluated for its production.

Hybrids that produce monoclonal receptor molecules that bind to protein molecule ligands The cells are then cultured to make additional quantities of the cells. Preferred implementation In addition, these cultured hybridoma cells also contain molecules that bind to polypeptides. It creates noclonal receptors.

Another embodiment of the invention is a kit for assessing the presence of cancer protein ligands. Provides a diagnostic system. This system consists of a first package containing a monoclonal receptor molecule of the invention. including at least A given amount of these receptors is evaluated for the presence of oncoprotein ligands. When mixed with a predetermined amount of the aqueous composition to be treated, the oncoprotein becomes bound by the receptor molecule. contains an amino acid residue sequence corresponding to the amino acid residue sequence of the polypeptide , form a receptor-ligand complex by immune reaction.

The presence of the complex is preferably indicated on the label contained in the second packaging of the system. more easily identified. Preferred oncoprotein ligand-containing aqueous compositions include cell extracts, ovine Contains water, urine and concentrated urine. Urine or urine concentrate is easily obtained by non-human means. and easily concentrated to enable the performance of the diagnostic tests described herein. cell conditioned by the extract and transformed cells. ed) medium is also a suitable aqueous composition containing the oncoprotein ligand.

Evaluation methods are another contemplated embodiment of the invention. Here cancer aspects Riga body samples to be evaluated for the presence of Concentrated urine is mixed with a liquid solution containing anti-cancer protein receptor molecules. The mixture formed is complex (immune complex: reaction product or immune reactant) is a cancer protein. sufficient to form a bond between the ligand and the receptor molecule (antigen-antibody complex). The presence of a time-maintained complex is then determined.

Whereas urine in its obtained or concentrated form is the composition to be evaluated, any Sources such as polyclonal, oligoclonal or monoclonal anti-cancer proteins Receptors also find use in the invention. Monoclonal antibodies of the invention should be evaluated Useful for other samples.

Determination of the presence of immunoreactants typically involves radioisotope- or enzyme-labeled Staphylococcal antibodies that bind to the receptors of the antibodies or immune complexes formed A particularly novel aspect of the present invention is that ureus protein A is used as a body sample. This is the use of urine. The evaluation described here uses concentrated urine as described. Implemented Vine. Cancer gene-related proteins have not been previously identified in urine samples. The evaluation aspect of the invention involves the identification of numerous oncoproteins that provide the mode of immunoreactivity of the specific sample being evaluated. This can be done by using linked polypeptide ligands. style obtained Comparison with modalities obtained from individuals with known symptoms yields a diagnosis.

A method of confirming the presence of a female fetus within the uterus is also contemplated. Here's the boiled food I got from my pregnant mother. A boiled, reduced volume, preferably concentrated urine sample is added from right to left and from the N-terminus. (i) LMEQCWAYEPGQRPSF and (if) described at the C-terminus A formula selected from the group consisting of YREQIKRVKDSDDVPMVLVGNKC and a receptor molecule that has immunoreacted with the polypeptide. Urine sample is pregnant Harvested between about 16 and about 20 weeks. The mixture is added to the receptor molecules present in the urine sample. for a sufficient period of time to immunoreact with existing oncoprotein ligands. special immune reactants The existence of is then evaluated. Immune reactants include receptor molecules and oncoprotein ligands approximately 40 kg for receptor molecules that are formed during and immunoreactive with the polypeptide. Dalton's 5-17% polyacrylamide gel and the above polypeptide. A relative molecular mass of approximately 55 kilodaltons for receptors that immunoreact with putido(yo). show. The presence of immunoreactants with any of these receptor molecules indicates that a female fetus is in the womb. Indicates that it exists. The receptor molecule is preferably monoclonal.

In another embodiment of the invention, the monoclonal receptor molecule is an oncoprotein ligand. forms the active binding moiety of an affinity adsorbent useful for binding and purifying molecules. child Here, the receptor is placed on a solid support that is chemically inert to the oncoprotein, such as agar or a cross-linked support. Combined with Hashi agar. The affinity adsorbent thus prepared is then used to bind protein ligators. The protein ligand is mixed into an aqueous composition containing the receptor-bound polypeptide. Reversible when the peptide has an amino acid residue sequence that corresponds to the amino acid residue sequence of the peptide. Forms a receptor-ligand complex. The compressor formed in this way The protein ligand is then dissociated to give the protein ligand in its pure form.

The present invention provides several benefits and advantages.

One benefit of the present invention is that epitopes contained in polypeptides of known amino acid residue sequences Another benefit of the present invention is that monoclonal receptor molecules that bind to the retro contained in the known amino acid residue sequence of the protein ligand encoded by the virus. A monoclonal receptor molecule can be created that binds to an epitope, where These protein ligands are not required to induce production of receptor molecules; be.

One of the advantages of the present invention is that immunogenic polypeptides of moderate length and protein ligand fractions can be A protein ligand in which a polypeptide partially corresponds to a child amino acid residue sequence This is a high-yield method for producing monoclonal receptors that bind to both molecules.

Another advantage of the present invention is that it contains monoclonal receptor molecules that can assess the presence of cancer proteins. This is the provision of diagnostic systems such as kits.

Another advantage of the present invention is that diagnostic methods can be achieved using body samples obtained by non-invasive means. This is the offer.

Another advantage of the present invention is that proteins of various molecular weights can be detected and the precise amount of protein expressed in vivo can be detected. The goal is to enable differential and highly accurate evaluation of cancer genes.

Another advantage of the present invention is that it allows prediction of fetal development or other growth conditions including neoplasia. The objective is to provide a non-invasive diagnostic method using maternal or individual urine.

Further benefits and advantages of the invention will become apparent to those skilled in the art from the following description and claims. That should be obvious.

Brief description of the drawing In the drawings forming part of this disclosure: Figure 1 shows the immunology for detecting the presence of 5T-FeSV~T-fes oncoprotein. 1 is a photograph of an autoradiograph illustrating a clinical evaluation. Feline sarcoma virus (ST- FeSV) and feline leukemia virus B (FeLV-B) yder-Theilen) strain Cell extracts from approximately 105 strains of MSTF cells (Sen et al. Acad Sai Ninisny, 80.1246-1250 (1983)) 5 Electrophoresis on ~17% polyacrylamide gel, then nitrocellulose sheet Moved to. The transferred proteins were then transferred to 310FO3 (lane 1) or 522006 (lane 1). hybridoma tissue culture called 2) or used as a negative control. was reacted with supernatant from an anti-influenza hemagglutinin hybridoma. Po This for lyacrylamide gel separation and subsequent transfer to nitrocellulose and visualization. The procedure is referred to below as the Western plot method. Protein visualization is a material described later. This was accomplished as described in the Materials and Methods section.

Figure 2 shows p85 (85 kilodal 85 Dalton) to detect the presence of the FeSV fusion protein. 1 is a photograph of an autoradiograph illustrating scientific evaluation. MSTF of about 2♂106 Cellular extracts of cells were electrophoresed on 5-17% polyacrylamide gels and then electrophoretically transferred to a piece of cellulose. This nitrocellulose piece is 810FO 3 (Lane A): P43D19 (Lane B), P42C10 (Lane C), P4 1=50 diluted hybridoma from a hybridoma called 4E11 (lane D). With each 5 ml of Brideoma culture supernatant or as a negative control. auscher) gp 70 protein receptor producing hybridoma R206BO 8 (Lane EX Naiman and Elder, Brok Natl Acad Sai Nini Snee, 77.4524-4528 (1980)).

Conjugation was performed using peroxidase-labeled rabies as described later in the Materials and Methods section. The cells were visualized by the addition of anti-mouse IgG. Marker “p8” on the left side of Figure 2 5-” is a 5T-FeSV point of Dalton at 85 encoded by the fes gene. Indicates the position of reprotin.

As can be seen from the protein in lane E, this approach is effective for monoclonal receptors of the present invention. Allows visualization of protein molecules that are not bound more specifically. Visible in lane E Subtracting the non-specifically bound protein from the protein visualized in lanes A-D, we get The only protein bound specifically was the p85 oncoprotein encoded by v-fes. to show that

Figure 3 shows the presence of 35p-labeled Fe5V fusion protein, designated p85. 1 is a photograph of an autoradiograph illustrating precipitation evaluation.

CCL64 mink cells (MSTF cells; lanes B and D), or FeLV-B and These cells (MSTF cells: lanes A and C) infected with It was labeled with Ikrokyuri's 32P for 2 hours.

The labeled cell extract was then mixed with 3 μl of goat anti-FeLV p15 antibody (lane A and B) or 50 μl of supernatant from cultured hybridoma 5IOF03 ( Lanes C and D). Immune complexes regulated in this way using Staphylococcus aureus bacteria expressing protein A. collected. The precipitated complex thus collected is washed and then applied to its component parts. Dissociated. The subsequent protein was reduced and denatured using a 5-17% polyacrylamide gel. analyzed under electrophoresis. Markers “p35-” and “pr35-” on the left of FIG. ” is a 85-dalton 5T-FeSV fusion protein encoded by the fes gene; and 65 show the migration position of Dalton's FeLV Gag precursor protein.

Figure 4 shows the amino acid residue sequence of (i) monkey meat called p28sis. Positions 139-155 of the predicted sequence of tumor virus transforming protein (Debarre et al., B. Roku, Natl, Akado, Sai, Ninisni, 80.731-735 (1983 ) corresponding to the synthetic polypeptide (hereinafter referred to as polypeptide (C) or number 1 13 and PDGF2 (73-89)), and (u) avian erythroblast virus. A synthetic polypeptide corresponding to residues 2 to 18 of the predicted amino acid residue sequence of cancer protein. Tido (Rushrou et al., Science, 216.1421-1423 (1982)) (hereinafter referred to as polypeptide (d) or number 131) 1 is a graph showing the immunoreactivity of oligoclonal antibodies obtained. Keyhole Limbe Synthetic polypeptides conjugated to hemocyanin (KLH) are It was used to immunize mice as generally described in Section.

To test the specificity of oligoclonal antibody-containing sera prepared in this way 250 nanograms of unconjugated polypeptide or 500 nanograms of KLH It dries at the bottom of the microtitration groove and contains Nyman, Elder, and monoclonal antibodies. D's and Tisel Products, Edited by Katz, CLC Press , Boca Raton, Florida, pp. 23-51 (1982). Fixed with nol. The remaining part of the pool was diluted with 3% bovine serum albumin (BSA). Block against non-specific protein adsorption using an incubation time of 4 hours at 7°C. I checked.

Tissue culture medium supplemented with 10% fetal bovine serum in each well of the microtiter plate. Each 25-fold dilution of immunized mouse serum starting from a 1:400 dilution using μj was added dropwise and BSA was blocked.

After 10 washes with distilled water, the phosphate buffer was diluted 1:+OO with 1% BSA. Rabbit anti-mouse kappa antibody (synthon bionicin) in saline solution (PBS) Add 25 µl of Co., Ltd. (Kuss Inc., Kensington, Maryland) and incubate at 37°C for 2 hours. Incubated for a while. After another 10 washes with distilled water, glucose oxidase Goat anti-rabbit Ig conjugated to 1:500 diluted in 1% BSA in PBS 25 μl of G was added and incubated for 1 hour at 37°C.

The amount of glucose oxidase bound in this way is 1.2% of glucose. ABTS dye (Boehringer Mannheim) at 100 μg/ml in the presence of p 10Pg101 horse rub in 0.1 molar phosphate buffer with H6,0 Determined by adding 50 μl of a solution containing Escherichia peroxidase.

The optical density of the solution prepared in this way was determined using a Titterchik Microscanner. (Flo Laboratories Inc., Englewood, Calnarnia). Read at 414 nm.

Oligocouloin in serum generated against polypeptides related to sis and myb Binding exhibited by null antibodies is indicated by open and filled symbols, respectively. Ru. The antibody antigen is sis-related polypeptide (C) (0, O); myb-related polypeptide (C) (0, O); Peptide (d) (, ), and KLH (, ).

Figure 5 shows oligoclonal cells induced by synthetic polypeptides (C) and (d). Mouse antiserum containing receptor antibodies (receptor) was used as a probe to Immunological assessment to detect the presence of derived platelet-derived growth factor (PDGF) This is a photograph of an autoradiograph showing. PDGF extract is available in the Materials and Methods section. It was purified from classical platelets as described in .

Approximately 2.5 units of purified PDGF extract from platelets was added to 0.5% dodecyl sulfate. Minimal volume of solution containing sodium (SDS) and 5% 2-mercaptoethanol mixed with. The resulting mixture was boiled for 2 minutes, then 5-17% polyacrylamide Electrophoresed on gel. The proteins were then electrophoretically transferred to nitrocellulose [Naima N and Elder, Vairoron, 123.187-205 (1982) Ko, U. Following the Oester plot method, the strips were then cut.

The nitrocellulose pieces prepared in this way are then processed to prevent non-adherent protein binding. 3% BSA, 0.1% polyoxyethylene (9) octyl fluoride in PBS to phenyl ether (Triton(TM)) X-100, Rohm & Haascampa Nee, Philadelphia). Mouse diluted 1.200 4 ml of antiserum was then incubated on a piece of nitrocellulose.

After washing three times with a solution of 0.1% Triton(TM) X-100 in PBS, nitro Cellulose pieces labeled with 1251 Staphylococcus aureus protein A ( lanes 2 and 3) at 106 counts per minute or conjugated with peroxidase. 1:10 of goat anti-mouse serum (Tabo Inc., Burlingham, CA) 0.00 dilution and again in PBS with 0.1% Triton(TM) Washed with -100. The peroxidase conjugate has a pH value of 7.4. 0.0009% H,O□, 0.0025% 3,3°-dimethoxy in solution IQml Cybenzidine dihydrochloride (Eastman-Kodak Company, Rotinister, New York). Place the 125I labeled piece on a black net. Kusu Hi-Plus (Ei, DuPont de Nemois & Co. 9. XRP-1 film (Eatman-Kodada) (Co., Ltd., Rochinister, New York) and screened for 48 hours at -70°C. The image was developed by applying additional pressure to the image.

Lane 1 contains total protein stained with amido black. Purified platelet extract sis-related polypeptide (C) (lanes 2 and 4) or as a negative control. Promoted with antiserum raised against myb-related polypeptide (d) (lanes 3 and 5). displayed. BSA, ovalbumin, chymotrypsinogen or β-lac External molecular weight standards based on toglobulin are shown on the left.

Figure 6 shows the presence of PDGF using the same Western plot method as described above. 1 is a photograph of an autoradiograph to illustrate immunological evaluation for clinical trial. P DGF is described in Nyman, Nature, 307.180-183 (1984). 10% 2-mercaptoethanol before electrophoretic protein separation according to the procedure The samples were boiled in the presence (lanes A-F) or absence (lanes G-L) of chlorine. PDG 12 amino acid residues at the amine terminal of F-1 (referred to as PDGF-1(1-12)) Two oligoclonal antibody-containing antisera induced by and lanes B and H. Polypeptide from the central part of PDGF-2 [PDG F-2 (73-89) and polypeptide (c)] (which is called p28sis (corresponding to the amino acid residue sequence at positions 139-155) Clonal antibody-containing antisera were used in lanes D and J and lanes E and K. PDGF -2 at the amino terminal 17 residues [referred to as PDGF-2(1-18)] and cartilage. 20 residues of PDGF-2 located 36-16 residues from the boxy terminus [PDGF -2(126-145)], which corresponds to p28sis at positions 191-210. Antiserum containing oligoclonal antibodies elicited by Lanes C and 1, and Lanes F and 1 were used. Antibodies bound to proteins are Rabbit anti-mouse I as described in Mann (supra) and in the Materials and Methods section below. Using gG+, next 11! Staphylococcus aureus protea labeled with ■ Visualized with 1.0' cpm of fin A.

70.00 in three cell lines using the Unistern plot procedure. 1 is a photograph of an autoradiograph showing immunological evaluation of the presence of 0 Dalton protein.

SSv-transformed NIH3T3 cells (lanes A-E), TRDI cells (natural transformed Ba1b/3T3 cell line) (lanes F-J) and MSTF Cells [productively infected with Snyder siren strains of FeLV-B and Fe5V] Approximately per lane from each of the mink lung line (CCL64) (10 per lane) Extracts from 106 cells were transferred to nitrocellulose sheets and treated with Unistern Pro. It was subjected to the cut method. Oligoclonal induced by PDGF-1(1-12) Antibody-containing antisera were used in lanes A-CSF-H and -M. PDGF -2(73-89)-containing oligoclonal antibody-containing antiserum NDXE, LJ.

Used for N and ○. Antiserum is used before immunoreaction with transferred cell extracts. , 100 micrograms of polypeptide PDGF-1(1-12) (lane A, D, F, L K and N), PDGF-2 (1-18X lanes B, G and L) and PDGF-2 (73-89) (lanes C, E, H, J, M and O). Incubated. Proteins were visualized as described in Figure 6.

Figure 8 shows 5SV-deformed normal rat kidney and normal rat kidney (NRK) cells. Immunological analysis of the presence of p2Qsis in culture media conditioned by A photograph of an autoradiograph showing the evaluation. 25ml non-concentrated medium equivalent to 5Sv-transformed cells (lanes A, CSE and G) or NRK cells ( Proteins from enriched media conditioned by lanes B, D, R and H) were transferred to nitrocellulose and then subjected to Western blotting. ) (lanes A-D) and induced by PDGF-2 (73-89X lanes E-H). and mixed with oligoclonal antibody-containing antiserum. Serum contains transferred proteins. 100 milligrams of the polypeptide PDGF-2 (73-8 9X lanes A, BSG and H) and PDGF-2 (1-18X lanes C, D SE and F). The immune response was as described in Figure 6. visualized. The marker p20sis on the left side of Fig. 8 is the position of p20sis. shows.

FIG. 9 shows the urinary sis and fes antisera from human cancer patients. Here is an autoradiogram to illustrate the immunological evaluation of the presence of proteins associated with this. This is a photo of F. The liquid body samples in this evaluation are described in the Materials and Methods section. This was the urine concentrate obtained in this manner. Concentrated urine is 5-17% polyacrylic electrophoresed on a Lumid gel and then on nitrocellulose.

Urine from three donors was concentrated 200 times, dialyzed, and 20 μl of each concentrate was The proteins were electrophoresed and transferred to nitrocellulose as described above. these three Human donors were diagnosed with rectal cancer (lanes A1D, G and J), liver cancer (lanes B, E, H and K), and Elaine sarcoma (Ewing's sarcoma) (lanes C, F, I and L) . sis-related polypeptide PD brain-incubated with immunizing polypeptide GF-2(73-89)-induced oligoclonal receptor-containing antiserum used in lanes D-F, while the v-fesST oncoprotein at positions 744-759 The same antibody was incubated with a fes-related polypeptide corresponding to the sequence Serum was used in lanes A-C. Similarly, incubate with the immunizing polypeptide. The oligoclonal receptor induced by the above fes-related polypeptide Containing antiserum was used in lanes G-1, while the above-mentioned sis-related polypeptide The same incubated antiserum was used in lanes JL. Oligoclonal receptor The immune reaction (binding) between the body and the protein can be performed as described for Figure 6. Visualized. The locations of sis and fes related proteins detected in urine concentrates are as follows: and at the right end by markers "Sis" and "fes", respectively.

Figure 10 is an autoradiograph showing immunological evaluation of the presence of ras-related proteins in urine. This is a rough photo.

Urine was tested at 250x (lanes A and B), 35x (lane C), and 70x (lane D). ), 75-fold (lane E) and 325-fold (lane F). Dialyze the urine Twenty microliters of each concentrate was electrophoresed and the proteins therein were nitrated as described above. Transferred to cellulose. The donor is normal (lanes ASB and F) or below. Conditions, 38 weeks gestation (lane C), lymphoma (lane D) and colon cancer (Lane E). The same normal patient was tested at 14-day intervals. Provided the urine samples collected and used in lanes A, B and F.

Whole urine samples were collected from residues 744-759 of the polypeptide fesST (lane A), Residues 96-118 of peptide rasHa (lane B) or polypeptide c-s p21r incubated with residues 138-154 of is (lanes C-F) 10 microliters induced at as (polypeptide 142) residues 96-118. was evaluated using anti-raslli water.

(Immunoreaction and binding between oligoclonals and proteins) is shown in Figure 6. Visualized as mentioned. The location of ras-related proteins detected in urine concentrate is It is shown in the left-hand side carry by the marker ras.

The protein detected to be associated with the ras oncogene is a ras-related polypeptide. ATCC number raised to 142, hybrid named HB8679 detected by monoclonal antibodies secreted by the tumor. This is about 55 protein is detected in lane A, and its activity is compared with the immunopeptide (lane B). blocked by incubation with collected from the same normal individual. Urine contains protein after 2 weeks (lane F). This protein was tested in pregnant patients (lane C) and from the urine of cancer patients (lanes D and E).

Figure 11 shows the data on 23 of the three cell lines using the Unistern plot procedure. Figure 2 is a photograph of an autoradiograph showing immunological assessment of the presence of Luton protein. drawing lanes are mink lung line sarcoma virus (MSTF) cells (lanes A-F) mink lung cell lines transformed by the Snyder-Schielen strain or is approximately 106 cells per lane from the uninfected MSTF cell line CCL (lanes GL). Contains extracts from cells. Each cell extract was extracted from a polyacrylamide gel using nitro Transferred onto a cellulose sheet and then subjected to Western plotting means.

The extract contains residues 5-16 of v-rasHA (ras-2, lanes A, G). Corresponding polypeptide 141 or p21 ras (ras-1, lane B, H) was incubated with polypeptide 142 corresponding to residues 96-118 of corresponding to residues 96-118 of p21 (ras-1, lanes A, B, G, H) Antiserum raised to polypeptide 142 was used for evaluation.

The same cell extract was added to the remaining p85-fes (fes-1, lanes CSD, LJ). Group 744-759 or p85-fes (fes-2, Le: /E, F.

K, L) raised to polypeptide 121 corresponding to residues 744-759 of It is evaluated as clear. The antiserum is fes-prepared before immunoreacting with the transferred cell extract. 1 polypeptide (lanes D, J), fes-2 polypeptide 744-759 (lanes D, J), lanes F, L) or ras-1 polypeptide (lanes C, E, LK) and brain Curated. Proteins were visualized as described for Figure 6.

Figure 12 shows the naturally transgenic mouse 3T3 cell line TRD-1 (lanes A and B). or the presence of secreted proteins in the supernatant from the human T-24 bladder cancer line (lanes C, D). 1 is a photograph of an autoradiograph showing the immunological evaluation of . The supernatant contains secreted fe The cell lines assessed for the presence of the protein were grown in the absence of serum and produced 4 Collected after 8 hours. 35 microliters, 1500: IT-24 cellular Polyacrylic acid concentrate or 1000:I TRD-1 cell concentrate Electrophoresis was performed in midgel and then transferred to nitrocellulose.

v-fesS corresponding to residues 744-759 of p85 fes (fes-2) Mouse antiserum against T synthetic polypeptide was used for evaluation. Antiserum is v-fes Synthetic ports corresponding to residues 519-530 of ST (feS1, lanes A and B) fes-2 polypeptides and brain curatives raised to repeptides or antisera. Beated.

The antiserum is then immunoreacted with the transferred cell supernatant. Proteins are shown in Figure 6. It is visualized as described in .

FIG. 13 shows ras-related proteins in cell extracts using Western plot means. 1 is a photograph of an autoradiograph showing immunological evaluation of the presence of .

Cell extracts of approximately 10 naturally transgenic 3T3 cells were used in lanes A-D. . 35 microliters of 48 hour supernatant from mouse 3T3 TRD-1 cells. A 1500x S condensate was used for lane EH. Proteins in the supernatant were collected using polyacrylamide gel. and then transferred onto nitrocellulose. v-fesHA Oligoclonal antibody against polypeptide 142 corresponding to residues 98-118 Containing antisera were used to detect unrelated fes polypeptides (lanes AXC, E, G) or immune ( Brain incubation with ras polypeptide used for lanes BSD, F, H) be done. Proteins were visualized as described in FIG.

Figure 14 shows the relationship between ras and si in cell extracts using Western plot means. Photograph of an autoradiograph showing immunological evaluation of the presence of s or fes related proteins. It is. The lanes in the drawing are for feline sarcoma virus (MSF cells). Approximately 106 cells per lane of mink lung cells transformed with Snyder Schielen strain Contains extracts from

The extract was isolated from residues 96-118 of p21ras (polypeptide 142, lane 2). , residues 1-18 and v-f of PDGF-2 (polypeptide 112, lane 1) The polypeptide corresponding to residues 744-759 of es (polypeptide 127, lane 3) was evaluated using an antiserum raised to ptide. Proteins are described in Figure 6. It was visualized as follows.

FIG. 15 shows the urinary s encoded by or related to is, fes and ras oncogenes. 1 is a photograph of an autoradiograph showing immunological evaluation of the presence of various proteins associated with Book Solution samples in the evaluation were obtained as described in the Materials and Methods section. It was a urine concentrate. The concentrated urine was electrophoresed on a 5-17% polyacrylamide gel. and then electrophoresed on nitrocellulose.

Urine from eight donors was concentrated 40 times, dialyzed, and 25 microliters ( (equivalent to 1 ml of unconcentrated urine) was electrophoresed, and the proteins therein were analyzed as previously described. was transferred to nitrocellulose. These donors had multiple bone marrow (lane 1, panel lanes A and B) gastric cancer (lane 2, panels A and B); lane 1, panels C and D; ) 35 weeks gestation (lane 3, panels A and B) lymphoma (lane 4, vein (p ane) A and B), gastric cancer (lane 1, veins C and D), 36 weeks gestation (lane 1, veins C and D), lane 2, panels C and D), breast cancer (lane 3, panels C and D) 39 weeks gestation (Lane 4, Panel C end D) and breast cancer (Panel E).

5is (panels A and B) ras (panels C and D) or fes related ports]) .

Monoclonal or oligoclonal receptors induced by peptide (panel E) Contained antiserum is used to probe each sample to assess immune polypeptides. used. 20 microliters of ascites (induced by hybridoma ATCCHB) and corresponding to positions 1-18 in the sequence of PDGF-2. sis-related polypeptides induced by hybridomas raised up to 112. panes C and Dl and A and B) or mouse plasma (fes The polypeptide corresponding to positions 744-759 in the oncoprotein sequence was raised. Panel E) contains 100 micrograms of immune ras polypeptide 142 (panel A). SD, and lane 2 of panel E), sis polypeptide 112 (panel B and C) or fes polypeptide (panel E rune 3) and erbB (panel E rune 3). The polypeptide corresponding to positions 366-381 written out in the cipher by rune 3) or corresponding to positions 590-605 of abl (panel E rune 4). Pre-incubated with polypeptide 312 for 30 minutes at 37°C. Following vation, samples were diluted with 3% BS in PBS at a pH of 7.4 ([3% BS A, oJ1 Vercented υton (trademark) X was uniformly evaluated. The connection is shown in Figure 6. Visualization was performed as described in .

Figure 16 shows immunological evaluation of the presence of ras, sis and fes related proteins in urine. This is a photograph of an autoradiograph showing the valence.

Urine was collected at monthly intervals from donors previously diagnosed as having active breast cancer. (lane 1.7.2.5.8.3.6.9, panel A). Collect urine and dialyze it Equivalent volumes of , 1 mI unconcentrated urine were applied to each lane in panel A.

In panel B, a portion of the same sample used in panel A, lanes 3.6 or 9, c1ooo Microliter Rune 1) applied to the following equivalent amount of unconcentrated urine, 500 microliter (lane 2), 250 microliter (lane 3), 125 microliter Lolitre (lane 4), 60 microliter (lane 5), 30 microliter (lane 6), 15 microliters (lane 7), 75 microliters (lane 7), 8).

The sample was ras (positions 96-118, polypeptide 142, panel A, lane 1 -3, panel B), fes (positions 744-759, polypeptide 127, panel A rune 4-6) or sis polypeptide (PDGF-2 positions 1-18, polypeptide peptide 112, panel, lanes 7-9), synthetic peptide and brain cube Prepare and probe as described in Figure 15, except without performing the Ta.

Figure 17 shows immunological evaluation of the presence of ras and fes related proteins in urine. This is a photo of a radiograph. The donor of the urine sample being evaluated was diagnosed with recurrent breast cancer (recurrent breast cancer). Lanes 1.2) or normal individuals (lanes 3-8).

Evaluation of ras-related proteins (panel A) and fes-related proteins (panel B) was performed in the first It was carried out as described in Figure 6. Samples evaluated were from breast cancer (lane 3) A patient in clinical remission, and the same patient 3 months later when the breast cancer reappeared (lane 2). samples were collected every 3 days and from normal females (lanes 3-5). Normal females should be collected every 12 hours (lanes 6-7) and normal males (lane 8). there were.

Figure 18 shows ras, fes and Sis in urine samples from donors with cancer. Figure 2 is a photograph of an autoradiograph showing detection of related proteins. Breast cancer (lane 1), before Prostatic carcinoma (lane 2), prostatic nodules! ri (lane 3) or lymphoma (lane 4) Urine from donors of 5is (Panel A ), ras (panel B) or fes (panel C) probed using antiserum. Less than p56s in lane 1.2 Slow moving horizontal stripes indicate excess albumin in these samples. . Increased levels of p"sis and p"sis during panel A rune 1.2 from donors with breast or prostate cancer, although associated with Other urine samples in the absence of elevated albumin levels (data not shown) The slowest in panel B, lanes 1-3, contains increasing levels of sis-related proteins. For moving horizontal stripes, check pIOOras. On the other hand, in panel B lane 1-4 A slightly faster horizontal stripe than the L chain of p''ras confirms p''ras.

Figure 19 is an autoradio showing detection of cancer gene-related proteins in urine from gestational donors. This is a photo of Ograph.

Four urine samples from the same individual collected one week apart during the last month of pregnancy were Related polypeptide 112 (PDGF-2 positions 1-18, panel A), ras polypeptide Peptide 142 (positions 96-118, panel B) or fes polypeptide 127 (positions 744-759, panel C). Panel C Overexposure was associated with the presence of p”fes (lanes 3 and 4) and p”fes (lane 4). Indicates presence. The light chain moves slightly faster than the horizontal stripes or at the bottom of the gel (panel C). -2-4) protein was detected with mouse antiserum against tes peptide. Canada In addition, a 150,000 Dalton protein was also detected using a mouse antiserum against the fes peptide. was detected in Urine samples were collected at weekly intervals.

Figures 20.21 and 22 show three oncoproteins with protein kinase activity. It is a table showing the amino acid sequences of conserved parts. These parts are part 20.21 and 22, named as the conserved kinase moieties, respectively. Oncogenes that program oncoproteins with kinase activity are listed in the left-hand column. Indicated by ordinary symbols. The middle column shows conserved amino acid residues starting from the N-terminus. Confirm the position of the base sequence in the oncoprotein polypeptide sequence. The right-hand column is from the left To the right, and from the N-terminus to the C-terminus, the amino acid residues of these conserved parts The base sequence is shown. Amino acid residue sequences also induce the production of monoclonal receptors of the invention. A sequence of polypeptides useful as a primary immunogen.

Figure 23 shows 51 control (normal donor) urine samples and various malignancies. Figure 2 is a table showing the frequency of detection of cancer gene-related proteins in urine samples from donors. in urine The amount of cancer gene-related proteins was evaluated using immunoplot method and divided into four categories. , undetectable, detectable, 5 to 15 times increase and greater than 15 times increase. there was.

p”ras represents approximately 70% of all samples from donors with neoplastic disease. was detected on However, similar frequencies are found in apparently normal individuals. Ta. The most striking elevation of p2Iras was in uterine and gastric cancers, as well as mini-roma. and in samples from donors with molar pregnancies, all of which This was a greater than 15-fold increase in protein to at least 30% of the protein.

Figure 24 shows various levels of cancer gene-related proteins in 260 urine samples from gestational donors. Figure 2 is a table of data reflecting the detection of files. The samples are graphed according to the third trimester of pregnancy. I divided it. Multiplex urine collections were obtained from multiplex donors. Evaluation included the following materials and and the procedures described in the Methods section. Donation with breast cancer As discussed below, very high levels of p”ras was detected in the gestational donor group throughout the course of pregnancy. sis and fes Associated proteins increased with progression of pregnancy.

Levels of pHras changed dramatically during several stages of pregnancy. symmetrically, p5 Levels of 5ras were detected in multiple samples from normal or breast cancer donors, and ras-related Protein concentrations increased significantly by more than 15 times per week in some donors.

The concentrations of the three sis-related proteins remained almost the same throughout the last month of pregnancy.

p35fes is detected during the last two weeks of pregnancy, while p”fes is the last week of pregnancy. was detected on the same day.

Urine samples collected 6 weeks postpartum contain elevated concentrations of these sis-associated proteins. However, ras and fes-related proteins returned to normal (data not shown). .

Figure 25 shows an immunoplot of mink lung cells transformed with fes. Mi skin cell extracts are either fes (level A-1) or erbB (level, J, K). probed with various antibodies against.

Figure 26 shows an immunoplot of human epidermoid carcinoma cells. human epidermoid carcinoma cellulite Extracts of mint were probed with the same antibodies used in Figure 1.

Figure 27 shows an immunoplot of a concentrated urine sample from a gestational diabetic patient. pregnancy sugar Concentrated urine samples from urinary patients were tested for antibodies used in Figure 1. Figure 28 shows endometrial tumor extracts. An immunoplot of the sample is shown. Extract of endometrial tumor (N.I.H. is the serial number) No. 071-781473-1), fes (lanes A-H) or erbB (lanes The probe was probed with an antibody against the I-L). Antibodies in lanes A-D are ELISA assays. gives a reactivity pattern different in value from those of lanes E-H. Lane I and J are directed to the region of v-erb B, and to the lane and L are resulting in different reactivity patterns for the same oncogene.

Figure 29 shows an immunoplot of breast sarcoma extract. Breast sarcoma (NIH acceptance number) No. 121-960-1) metastasizes to the lymph nodes. However, the extract was probed with the antibodies used in Figure 4.

Figure 30 shows the immunoprocedure of breast sarcoma (NIH receipt number 3l-14459). The lots are shown and the extracts were probed with the antibodies used in FIG.

Figure 31 shows the immunotherapy for uterine sarcoma (NIH receipt number 3l-13530). The plot is shown and the extract was probed with the antibodies used in Figure 4.

Figure 32 shows breast sarcoma (NIH accession number 121-960-1), which is a lymphoid tumor. An immunoplot of L/Taga metastasizes to a node is shown.

This metastatic breast sarcoma extract contains ros (lane A), fes (lane B), BTG F (lanes C-G), ras (lanes H-J) and erbB (lanes-N) probed with an antibody against.

Figure 33 shows the immunohistochemistry of metastatic uterine cancer derived from NIH sarcoma 31-18265. Show the plot. This cancer had metastasized to the omentum and was probed with the antibody used in Figure 8. .

Figure 34 shows metastatic colon cancer (N2H entry number 3l-18152), which is a Iminoplot showing metastasis to lymph nodes. This extract is the anti-inflammatory agent used in Figure 8. I probed it with my body.

Figure 35 is an illustration of metastatic uterine cancer (N2H receipt number 031-10128-1). A munoplot is shown. Uterine cancer metastasized to the omentum, but this extract Probed with the antibody used in the figure.

Figure 36 shows lymphoma from the pancreas (N2H receipt number 021-50073-1). ) is shown. This extract was probed with the antibodies used in FIG.

Figure 37 shows the illustration of breast cancer extract (NIH receipt number 031-1239-1). A munoplot is shown. This extract was probed with the antibodies used in FIG.

Figure 38 is an illustration of rectal sarcoma extract (N2H receipt number 031-1239-1). A munoplot is shown. This extract was probed with the antibodies used in FIG.

Figure 39 shows the immobilization of metastatic lung cancer (N2H receipt number 041-78297-1). No plot is shown. This lung cancer has metastasized to the lymph nodes, and this extract is Probed with the antibody used in the figure.

Figure 40 shows an immunoplot of rat striatum. Rat striatum extract H /N-RAS (Lane A), H-RAS (Lane B), MMC (Lane C), v -myb (lane D), 1nt-1 (lane E) and two different sis inductions probed with antibodies (lanes F and G).

Figures 41 and 42 show meat derived from cell lines deposited with the NIH depository. 1 is a table showing reactivity patterns of tumor extracts. Extracts were probed with various antibodies. , the pattern of reaction results was scored for the presence and level of oncogene products. profit Points were based on the strength of horizontal stripes derived using immunoplot techniques.

Figure 43 shows the synchronization of urinary cancer gene-related proteins in pregnant trophoblast patients undergoing chemotherapy. Indicates asynchronous appearance. chemotherapy Serial urine samples from pregnant trophoblastic patients undergoing the method showed sis residues (lane A). H/N-RAS residues (lane C), MYC residues (lanes D and E), src residues (lanes F and G) and 1nt-1 residue (lane H). probed.

Figure 44(A). Immunoplot of normal, healthy control serum. 100rrl blood A sample of the serum was incubated with antibodies against peptide sequences predicted by the following oncogenes. Lobed: 5is (lane 1), fes (lanes 2.3 and 15), β-T GF (lanes 4 and 5), 1nt-1 (lanes 6 and 12), myb (lane 7) ), 5rc (lane 8), c myb (lanes 9.13 and 14), mos (L / -n 10), and )(-ras (lanes 11 and 16).

Figure 44(B). with multiple carcinogen exposures (PCBs, asbestos, smoking); Immunoplots of serum from individuals without clinically detectable malignancy. 100μt A sample of serum was added to the peptide sequence predicted by the oncogene defined in Figure 1(A). probed with antibodies against the column.

Noticeably lower in lanes 11 and 16, not seen in normal, healthy controls. Please pay attention to the obi. These bands are part of the Harvey ras tumor. Corresponds to gene-encoded P21.

Figure 45. With multiple exposures to carcinogens, (a) pre-clinical development of colonic polyps18 (b) Immunoplot of patient serum 6 weeks after polyp removal. (b) to Note the presence of the ras-encoded P-21 protein in (a). sea bream.

Detailed description of the invention The present invention provides monoclonal receptor molecules for all cancer protein ligands, general methods of inducing or producing the body, and products and methods using these receptors. related. Terms often used herein are defined below: Receptor - A biologically active molecule that binds to a receptor and to a ligand. of the present invention The receptor molecule can be a complete whole antibody or a near-complete antibody or an idiotype of the antibody. It is a containing polyamide part. The biological activity of the receptor molecule is determined by at least the biological p Receptor binding to antigenic ligand when mixed in aqueous medium at H value and ionic strength It is proven by Preferably the receptor also has a pH value range of about 5 to about 9. and the ionic strength of distilled water to the ionic strength of IM sodium chloride. binds to the antigen ligand with high strength.

The idiotype-containing polypeptide portion of an antibody (antibody binding site) an antibody containing the Fab and (ab)2 portions of the antibody; body molecules, which are well known in the art and which are used to form whole antibodies, respectively, papain and peb. It is produced by a well-known reaction method. For example, Thiophylopoulus and Dikiran See U.S. Pat. No. 4,342,566. Whole antibodies are preferred , is used as an example of a receptor molecule considered in the present invention.

Monoclonal receptors - Monoclonal receptors (Mabs) are the only type of receptor. Produced by single-cell clones called hybridomas that secrete only offspring It is a receptor. Hybridoma cells are antibody-producing cells and myeloma or other self-perpetuating cells. fused from cell lines that Such receptors were first discovered in cola and milste Described by John, Nature, 56.495-497 (1975), this article The statement is incorporated herein by reference.

Oligoclonal receptors - Oligoclonal receptors are of moderate length, e.g. on a polypeptide of 7 to about 40, more preferably about 10 to about 30 amino acid residues. A receptor that is triggered by and binds to one or more epitopes. Oligoku Local receptors are usually a mixture of receptors produced by - or more cells.

Oligoclonal receptors so created are usually composed of protein chain(s). Polyclonal polyclonal molecule generated for all protein molecules that can have shrimp-dove regions throughout their length. It is more epi-dob specific in its binding than the null receptor. useful here Animals immunized with polypeptides containing oligoclonal receptors (antibodies) Make serum.

Ligand - Ligand is the protein or polypeptide to which the receptor of the invention binds. It is a peptide.

Corresponding (or equivalent) - The word corresponding or equivalent refers to the amino acid residue sequence. when used in conjunction with a sequence, the amino acid residue sequence of the first polypeptide or protein; is sufficiently similar to the amino acid residue sequence contained in the second polypeptide or protein. and therefore the receptors raised for the first one (e.g. antigen-synthesizing polypeptide) is immunologically linked when mixed in an aqueous composition with a second substance (e.g., an oncoprotein). It means that it matches. Such polypeptides and/or proteins may It also contains cognate epitopes and therefore contains at least about 6 to about 8, e.g. It can be said that they share a similar sequence of residues.

epitope-containing amino acid residue sequences of corresponding first and second polypeptides or proteins; The columns are most preferably identical. However, amino acid residues within the epitope changes, preferably conservative changes, and deletions or additions of residues; A well-known cross-linking reaction between a receptor for one polypeptide or protein and a second It will still be possible to respond. Conservative changes in amino acid residues are well known and include lysine ( Between Lys:k) and arginine (Arg;R), aspartic acid (Asp;D) and glutamic acid (Glu; E), between leucine (Leu; L) and isoleucine ( Residue exchanges such as between I le; I) are included.

Polypeptides useful in the present invention include amino acids corresponding to part of the amino acid residue sequence of proteins. It is often described as having a sequence of amino acid residues. Such polypeptides are preferably is a Cys residue used to bind or link the polypeptide to the carrier It contains only the terminal residues such as, plus the identifyingly corresponding amino acid residues. in protein Additional amino acid residues that do not correspond to residues may be present at the polypeptide terminus; However, the use of such residues, although contemplated in the present invention, is usually wasteful and Undesirable.

Similarly, a protein is a polypeptide whose amino acid residue sequence corresponds to a portion of it. It is described as having an amino acid residue sequence of This term refers to polypeptides and proteins. It is intended to show the same relationship as above.

The full name of an individual amino acid residue is sometimes used as well as the well-known three-letter abbreviation. It will be done. Single letter symbols for amino acid residues are most often used. Correspondence below The table shows the full name of each amino acid residue listed here, as well as abbreviations and symbols. show.

Amino acid three-letter abbreviation - letter symbol Asparagine Asn N Aspartic acid Asp D Asparagine + Aspartic acid Asx B Glutamic acid Glu E Glutamine + Glutamic acid C1x Z glycidi G1yG Histidine His H Methionine Met M Phenylalanine Phe F Proline Pro P Serin Ser S Threonine Thr T Tributophone Trp W (N. L. Lehninger, Biochemistry; Wirth Publisher N. , Y7. N.Y., 1970) 1. Production of monoclonal receptors As mentioned above, the present invention provides for the use of residues of moderate length, such as from about 7 to about 40 residues, preferably Binds to an immunogenic polypeptide of about 10-30 residues, as well as amino acid residues thereof. A protein whose base sequence partially corresponds to the amino acid residue sequence of this polypeptide. Consider a monoclonal receptor molecule conjugated with a child ligand. Monochrome of the present invention The null receptor is an immunogenic polypeptide or a carrier-conjugated immunogenic polypeptide. generated or induced by the use of a conjugate; where the immunogenic polypeptide is a protein An amino acid residue sequence of moderate length that corresponds to a portion of the amino acid residue sequence of a molecular ligand. Contains columns.

Epitope localization of monoclonal antibodies has technical problems. whole bacterial genetics Monoclonal antibodies against the progeny come in two different types, natural or denatured. can be produced using an immunogen. The use of natural proteins allows for purification and subsequent epitope This is the most important technical problem associated with all genetic mapping. Using natural protein The main advantage of monoclonal antibodies is that they block the physiological function of the target protein. It's production.

Oncogene products made in bacteria are comparable to gene products synthesized in more advanced organisms. Typically, but not structurally identical to the product. Direct evidence of this difference is sis given by analysis of gene products.

In mammalian cells, p28sis is rapidly dissociated to p2Qsis. Generally, bacterial p28SiS is not dissociated and does not form dimers. Bakte Indirect evidence of differences between other oncogene products made in avian or avian cells is largely Monoclonal antibodies raised against enteric bacteria-produced protein products can be used in transformed avian cells. It binds to immunogens much more effectively than to proteins synthesized in cells (immunogens). This is given by the observation that although the epigens are denatured).

The sequence of various oncogenes is due to the emergence and segregation of additional monoclonal receptors. Additional portions of the proto-oncogene sequence that may be useful for additional peptide synthesis of It seems that this could provide a basis for confirmation.

Therefore, although purification of denatured proteins is technically relatively easy, the resulting antiserum is may recognize one residue (conformation) unique to the Teria gene product. . This observation has significant technical difficulties for epitope mapping studies.

An approach for defining antibody epitopes is to use partial proteolytic The expression of protein fragments or subgenomic fragments is utilized. protein flag epitope mapping using It was first shown by I. Ninisny 77.4524 (1980); Even when a fleeting digest is evaluated with a large panel of monoclonal antibodies, Only an approximate determination of the binding site could be made. i.e. protein fragment Even with immunization, the definition of the binding site is limited. Also, the area that interested me is While there is no guarantee that clonal antibodies will be induced, known Immunization with an appropriate polypeptide of amino acid residue sequence involves a well-defined region, That is, an antibody (receptor) that immunoreacts with a region corresponding to the sequence of the immunizing polypeptide. ) production guaranteed.

Epitope mapping shows that changes in epitopes due to one amino acid can be quite different. other studies suggest that it may generate reactivity within the epitope. indicates that cross-reactivity is obtained when - or two or more amino acid residues are different. vinegar. Furthermore, immunization of mice with the same synthetic polypeptide of the same strain resulted in This may result in different reactivities being detected.

Hybridomas made with synthetic polypeptides also show that recognition is largely conformationally independent. Because it is a monoclonal receptor that reacts with the complete whole protein under various reaction conditions, Build a body. Therefore, Western plots, dot plots, fixed cells and autologous tissue and cell extracts, body fluids such as amniotic fluid, and concentrated or intact urine. It can be evaluated in the same way as natural protein. Also, a known precisely defined address in epitalk The amino acid residues allow the isolation of antibodies that can distinguish between single amino acid changes and thus Provides a means of determining the significance of limited changes in conserved regions of the proteins involved give.

Monoclonal antibodies directed against synthetic polypeptides also localize sites of protein interaction. Provides a means of diagramming. Differential coprecipitation of molecules associated with pp60src was reported. This suggests the identification of regions of the src protein involved in such interactions. Ru.

That is, an immunogenic compound with a region defined by the sequence of the polypeptide to be immunized. The induction of a monoclonal antibody (receptor) that accompanies a mature immune response is A complex procedure for the isolation of cool sensation proteins or the identification of epitope sites of their oncoproteins. Conformation-independent oncogene production, ensuring no steps are required. create receptors that recognize the compound. All of this makes the use of such receptors different. This will be extended to research.

Animal host protection involves the use of immunogenic polypeptides as agents in vaccines. However, it has been shown that it is possible to produce hybridoma antibodies (Mabs) in high yield. The possibility of using such immunogenic polypeptides to produce I mentioned earlier that this was not considered possible. Each Mab produces a unique uniqueness Anti-polypeptide antibodies that recognize even complete protein molecules because they are derived from a single cell The ratio of the number of clones that make the polypeptide to the total number of polypeptides that recognize the clone is the polypeptide A reasonable estimate of the true conformational frequency of de can be given.

The results described here are contrary to the established model described above, and the moderate length used here is The frequency of these polypeptides was previously predicted, assuming a conformation similar to that of natural proteins. The Mab of the hybridoma was much higher than that of the synthetic polypropylene produced against it. The frequency of producing hybridomas that recognize both peptides and complete molecules is , about 4 orders of magnitude (about 10.000 times) larger than predicted by the probabilistic model. Traditional researchers have developed immunogen molecules to create antibodies that recognize these polypeptides. Ripeptides have been used for several decades. In addition, regarding the production of monoclonal antibodies, The Kolla and Milstein article cited above was published in 1975. this Year, 1975 onwards, Arnheiter et al., Nature (London) 294.27 8-280 (1981) used polypeptide immunogens to develop monoclonal antibodies. I have described an attempt to make one. As mentioned earlier, the results of Arnheiter et al. It requires the use of human liver from infected mice, and the large 56-mar polypeptide, such as and the only I that recognizes the protein whose polypeptide corresponds to the sequence of the protein. It should be considered a failure in that gC type monoclonal antibodies were obtained.

The immunogen polypeptide partially corresponds to the amino acid sequence of the immunogen and protein ligand. A model made from an immunogenic polypeptide that recognizes both protein ligands and protein ligands. There are at least two reasons why there are relatively few reports on the production of noclonal receptors. It is thought that it depends on the factors. First, a small amount, if any, of monoclonal antibodies. According to a probabilistic model that predicts that the product will not be produced. There is a dominant way of thinking. second Researchers such as Arnheiter et al. I once had no law. Because the present invention arose for polypeptides Monoclonal receptors are derived from monoclonal antibodies made against complete proteins. Although it is made in a different way.

That is, the amino acid residue sequences of the immunogenic polypeptide and protein ligand include this polypeptide. A 1g class that recognizes both proteins and cants to which lipeptides partially correspond. To successfully create a monoclonal receptor, follow the steps outlined below. Must.

Conjugation of the immunogenic polypeptide alone or linked to a carrier A body is created. The polypeptide has about 7 to about 40, preferably about 10 to about 30 It has amino acid residues of moderate length, such as the amino acid residue sequence of Immunogen polypeptide The amino acid residue sequence of the protein is the amino acid residue sequence of a protein molecule ligand such as a cancer protein. corresponds to a part of Although immunogenic polypeptides can be used alone as a Carriers such as iphorimbet hemocyanin (KLH), bovine serum albumin (BSA), albumins such as human serum albumin (BSA), blush cells and e.g. sheep red blood cells, tension toxoid and edstin, and It is bound to a polyamino acid such as poly(D-lysine=D-glutamic acid), etc. It is preferred to use a polypeptide as the conjugate.

Immunogenicity and antigenicity of polypeptides are determined by the use of polypeptides in keyhole limpets. The conjugate is bound to a mocyanin carrier, and the conjugate thus obtained is then used to produce a mouse. It can be tested by immunizing the body. The immunizing polypeptide or conjugate is , a physiologically acceptable diluent such as physiological saline, phosphate buffered saline or or dissolved or dispersed in others well known in the art. Adjuvants (described later) (Bant) may also be included in the conjugate used for immunization.

Useful polypeptides should be diluted at least 1:400 after three immunizations in one month. oligoclonal receptor-containing antihematin in mice immunized against polypeptides of to give a 50 percent binding titer of the It is sufficiently immunogenic and antigenic. Here each immunization has a small number (at least about 1 o 1.1 g 1 preferably as little as (at least about 501.tg) Complete Freund's adjuvant for one immunization, followed by alum adjuvant. use

Although this test need not be performed prior to use of a given polypeptide as an immunogen, , but see if the polypeptide is useful in creating the desired monoclonal receptor. It is preferred to do so as a preliminary screening procedure. preliminary screening and The polypeptides useful herein as immunogens, whether or not used as The immunizing agents described above are used to give the titers described above.

After delivery of the immunogenic polypeptide, mammals such as mice, rabbits, goats, horses, etc. The immunogenic polypeptide or conjugate of the polypeptide attached to the carrier immunized and the receptor molecule is at least 1:400 diluted against the polypeptide. Give hyperimmune serum where percent binding titers are indicated. i.e. polypeptide The same animal, e.g., a mouse, in which one wishes to pretest the immunogenicity of the Mab is It can be used to

It is particularly preferred to use the same animals for Mab induction as used in the preliminary test. Delicious. This preference is due to the fact that once the 50 percent binding titer mentioned above is achieved, , accidental laboratory accidents such as contamination of cell cultures or the contamination of these cultures. With the exception of destruction, the human liver of this animal can be used as a source of antibody-producing cells to generate the desired specific Because hybridomas are almost guaranteed to secrete monoclonal antibodies, Ru.

The immunization reg necessary to create a hyperimmune state imen), as is known, among other things (animal type, animal weight, polypep Immunization of titanide and carrier (if used), adjuvant (if used) stated that it is a function of the genicity and amount and number of immunizations administered at a given time. It is. above to obtain a 50 percent combined titer with at least a 1:400 dilution. The immunizing agent produces a hyperimmune state in test mice and does not cause hyperimmunity in other animals. It can be used as a proportional basis for inducing epidemics. Three immunizations Polypeptides that are not necessary, but useful, to create a hyperimmune state Three such immunizations within one month are sufficient to create that condition. Otherwise, the polypeptide may be a hybridoma of the invention and its monoclonal antibody. It is further stated that it is not sufficiently immunogenic for high yield production in the body.

Serum oligoclonal receptor content so produced in hyperimmunized animals The child also includes an immunogenic polypeptide to which the immunogenic polypeptide corresponds in amino acid residue sequence to a portion thereof. However, it binds to protein molecule ligands. The bond evaluation is described in the Materials and Methods section below. It can be stated. There is no need to use pure samples of protein molecule ligands in these evaluations. , rather cell extracts or tissue preparations such as microscope slides containing protein ligands. It is stated that it can be used.

Hyperimmunized animals are maintained; i.e., without further immunization, with fewer After administration of the immunization, make a combined titer of 50 percent with a 1:400 dilution. can be kept alive for about 30 days. In other words, the animal is first given a hyperimmune state. The immune system is immunized so that hyperimmunity is allowed to recede.

Reduction of binding effects typically takes from 1 to about 5 months in mice. of combined titre This reduction occurs in primed sprouts when the immunogen is reintroduced. It is thought that this corresponds to a period during which the cells are able to mount a vigorous reaction.

Such stimulation by intravenous injection using immunogenic polypeptides or conjugates thereof The immunization is administered to the animal after the maintenance period has been completed, e.g. at least 30 days after the last administration. available. Antibody-producing cells such as human liver cells or lymphocytes from promoted animals are Next, in order to create hybridoma cells, within about 3 to 5 days from the day of effect-promoting administration. is fused with a myeloma cell line from the same animal type (species). The effect is promoted by buds. These cells undergo oligoclonal maturation with near-optimal amounts of polypeptides. It is thought to stimulate the body to the point of secreting antibodies.

SP210-Ag14 (ATCC CRL 1581 ), hyboxanthin-thymidine (HAT) sensitive, myeloma cell line Preferred for use in fusion with visceral cells.

Details of using this HAT strain for fusion are described in the Materials and Methods section below. It will be done. Hybridoma cells are then used to reduce overgrowth by non-productive cells and feeder monolayer or to provide a selection method for cells that grow easily in in vitro conditions Cloned at limiting dilution without or requiring macrophages. death However, a single layer of such feeders may also be used.

The hybridoma cells created in this way are then subjected to a model that binds to protein molecule ligands. Assess the production (secretion) of noclonal receptor molecules. This ligand is A part of a protein to which an epidemiogenic polypeptide corresponds in amino acid residue sequence. Ru. Hybrids are then used to create monoclonal receptor molecules that bind to protein ligands. The doma cells are further cultured to obtain additional amounts of these hybridoma cells and protein molecules. Make monoclonal receptors secreted by these cells that bind to the ligand.

Typically such cultures are grown at limiting dilution, e.g. It is performed on approximately 1 cell.

In a preferred manner, the generated hybridoma cells also contain a polypeptide immunogen. We also evaluated the production of monoclonal receptor molecules that bind to protein ligands. valued. A monoclonal protein that binds both the immunogenic polypeptide and the protein ligand is then used. Where the hybridoma cells producing the local receptor molecules are preferably cultured. These cells are.

When a sample of protein molecule ligands is limited, the ability to bind to the immunogenic polypeptide Initial screen of hybridomas for secretion of monoclonal receptors It is convenient. Hybrids that exhibit positive binding to this polypeptide Macrone is then typically frozen for storage. then they are unzipped , and a large number of monoclonal receptors are produced from many different hybridoma cells. To ensure that truly monoclonal antibodies are produced, rather than subcloned by limiting dilution. This limiting dilution subclone culture is re-cultured. and typically without phagocytic bodies or macrophages (as these are not needed) It will be done.

The final hybridoma cells produced are the well-known cells for such cells. It can be cultured according to conventional in vitro tissue culture techniques. More preferably, hybrid Doma cells are similarly cultured in animals using well-known techniques and are monoclonal receptors. The body is obtained from the ascites fluid thus generated. Used for the development of ascites Animals are typically sent to the Scripps Clinic and Research Foundation. 129XBALB/c bred in a mouse house in La Jolla, California. It's a mouse. However, animals other than mice are not used for hybridoma preparation. If the animal species is used for ascites production.

As mentioned above, it is preferred that the myeloma cell line is from the same species as the recipient.

Therefore, mouse-mouse hybrids [Shulman et al., Nature, Changyudan, 26 9 (1978)), or a rat-rat hybrid [Galfa et al., Nature 277.131 (1979)] are typically used. It will be done. However, some rat-mouse hybrids are also hybridomas. It has been used successfully in the formation of Ronal Antibodies By Cell Fusion”, Antibodies A Article in ZA Tools, edited by Marcaronis et al., Joan Wiley & N's Publishing, p273 (1982)]. Myeloma suitable for use in the present invention includes: MPC-11 (ATCC CRL 167), P 3 x 63-Ag8.653 (ATCC CRL 1580), Sp-210-Ag14 (ATCC CRL 1 581), P3x63Ag80.1 (ATCCSRL 1597), Y3-Ag 1.2.3. (Collection Nationale de Culticore de Microol Deposited with Ganismus, Paris, France, No. l-078) and P3x63A g8 (ATCCTIB 9). Myeloma line Sp-210-Ag14 and P3x63-Ag8°653 are preferred for use in the present invention.

That is, according to the method of the present invention, a known predetermined epitope of a protein molecule, such as an oncoprotein, can be detected. production of relatively high yields of monoclonal receptors that bind to or immunoreact with is now possible. In addition, at least about 1:40 to the immunizing polypeptide Hyperimmune serum containing oligoclonal antibodies exhibiting a 50 percent binding titer of 0. Once made by a person skilled in the art, he or she can follow the steps described above and extract from a hyperimmunized animal. A human liver is removed and its antibody-producing cells are transferred to a myeloma lineage from the same animal type or strain. and the hybridomas produced from this fusion are Monoclonal that binds to immunizing polypeptides and corresponding proteins such as oncoproteins Secretion of the receptor is virtually guaranteed. Such a result was previously impossible. there were.

The methods described above generate monoclonal receptors for virtually any protein molecule ligand. Useful for producing secreting hybridomas. Such hybridomas and An example of a monoclonal receptor is one whose amino acid residue sequence is encoded by an oncogene. immunogenic polypeptides of moderate length corresponding to the amino acid residue sequences of cancer proteins It was created by Examples of oncogenes and useful immunogenic polypeptides are listed below. The amino terminus in the oncoprotein sequence to which the polypeptide corresponds is shown in The numerical position from the end is given in parentheses. Here, the apposition of these polypeptides is The amino acid residue sequence is considered from left to right and from the amino terminus to the carboxy terminus. and is represented by an equation selected from the group consisting of the equations shown in Table 1 below.

Table 1 114 v-sis RVTIRTVVRRPPKGKHRKC110cms is L~’5ARQGDPIPEELVE (1-16) 111 PDGF- I 5 IEEAV PAVCKT (1-12) 112 PDGF-2SLGSLT IAEPAMIAECKT113 PDGF-2RKIEIVRKKKPIFKK ATV114 PDGF-2RVTIRTVRVRRPPKGKHRKCT 121 v-fes I GRGNFGEVF 5G (519-530)T 125v-fes 5nvllSFGILLWETFSLGASPYPNLSN QQTRT 126 v-fes 5PYPNLSNQQTR (711-722)T 128V-CWAYEPGQR, PSF (748-759)T 129 v-fes LWETFSLGASPYPNLSNQQTR131 v -myb RRKVEQEGYPQESSKAG(2-18)a 141 v-ras KLVVVGARGVGK(5-16)a 146 v-ras VTLVREIRQHKLRKLNPPDESGPGC( 157-181)i 147 v-ras VTLvREIRQYRLKKISKF, EKTPGC( 157-180) 148 v-ras YREQLKRVKDSEDVPMVL VGNKCu 144 v-ras KLVVVGAVGVGK (5-16) 145 N-RA S KLVVVGAGGVGK (5-16) 231 N-RAS DGETCL LDILDTAGQEEY237N-RAS YTLVREIRQYRMKKL NSSDGTQGC (157-181) 233 H-RAS YKRMKKL NSSDGTQGC234 K-RAS AGPEAQRLPGLLK (-1 3to-1) 235-RAS CGDSLAARQGAGRR(180to- 167) 149 v-bas KLVVVGAKGVGK (5-16) 150 MYCAPSEI)IWKKFELLPTPPLSP151 MYCCDEEE NFYQQQQQSEL (25-40) 152 MYCPAPSEDIWKKF EL (43-55) 153 MYCLPTPPLSPSRR3GLC (56-7 0) 154MYCCDPDDETFIKNIIIQDC(1,17-133) 155 MYCC3TSSLYLQDLSAAASEC156MYCCASQD SSAFSPSSDSLLSSTESSP (208-231)1+7 MYCC TSPRSSDTEENVKRRT158 MYC5VQAEEQKLISEE DLLRKRR159 MYCLRKRREQLKHKLEQLRNSC160 MYC111QDCMWSGFSAA (128-141)182 N-MYCP PGEDIWKKFELLPTPPLSP183 N-MYCVILQDCMW SGFSAR (110-123) 184 N-MYC5LQAEEHQLLLE KEKLQARQ185 N-MYCLQARQQQLLKKI EHARTC 192 L-MYCAPSEDIWKKFELVPSPPTSP193 L-M YCI　IRRDCMWSGFSAR(110-123)161　v-mos　 LRRELSPSVDSR (4253) 162 v-mos RQASPPHI GGTY (260-271) 153v-mos TTREVPYSGEPQ (3 01312) 164 v-mos I IQSCWEARGLQRPSA165 v-mos LGSGGFGSVYKA (100-111) 168 v-mo s TLWQMTTREVPYSGPQYVQYA761 v-mos TLW QMTTREVPYSGEPQ￥〜’QY166 c-mos I IQSCW EARALQRPGA167 MOS VIQRCWRPSAAQRPSA76 2MOS TLWQMTTKQAPYSGERQHILY171v-erb B IMVKCWMIDADSRPKF172 v-erb B LGSGAFG TIYKG (138-149) 173v-erb B ENDTLVRKYAD ANAVCQ (23-39) 174v-erb B VWELMTFGSKPY DGIP, ASEIS175 neu IMVKCWMIDSECRPRF17 8 neu ~’WELMTFGAKPYDG I PARE I P179 neu LGSGAFGTVYKG (731-742) 176HER-I RR RHIVRKRTLRRLQERE177HER-I VWELMTFGSK PYDGIPASEIS107 v-src LLNPENPRG Ding FLVRE SETTKG208v-src TFVALYDYESRTETDLSFKKG ERL(85-108)C 202v-src LGQGCFGEVWMG (273-284)RA 203 v-src G55KSKPKDPSQRRRS206 SRCLMC QCWRKEPEERPTF (note 2) 211 v-fgr AMEQTWR LDPEERPTF212v-fgr LGTGCFGDVWLG (410-4 21) 213v-fgr L-DELISKGRVPYPGMNNREVL214 FGRL location ITKGRIPYPGMNKREVL215 FGRLNPGN PQGAFLIRESETTKG2211nt-I DYRRRGPGGPDW HWGGC(1,54-170) 2221nt-I LHNNEAGRTTVFS (200-212) 2231n t-I EPEDPAHKPPSPHDL (275-289) 2241nt-I RACNSSSPALDGCEL (313-327) 240 v -yes LMKLCWKKDPDERPT (778-792) 241 v-yes L location VTKGRVPYPGMVNREVL242 v-yes VFVALYDYE ARTTDDLSFKKGERF (369-393) 243 v-yes LL NPGNQRGIFLVRESETTKG250 v-mil LVADCLK KVREERPLF252 v-mil VLYELMTGELPYSHINN RDQI251v-raf IGSGSFGTVYRG (355-366)”2 60 v-raf LVADCVKKVKEERPLF261 v-raf V LYELMAGELPYAHINNRDQI253 RAF IGSGSFGT VYKG (355-366) 262, to-RAF LLTDCLKFQREE RPLF266 A-RAF VLYELMTGSLPYSHIGSRDQI2 54 PKS IG Ding GSFGTVFRG (25-36) 255 PKS VL YELMTGSLPYSHIGCRDQI256 PKS LLSDCLKFQ REERPLF270v-rel TLH8CWQQLYSPSPSA(,38 2-397) 701　PKCLGKGSFGKVMLA(344-355)702　PKCL YEMLAGQPPFDGEDEDELF703 PKCLMTKHPGKRL GCGPEGE711 PKCLGKGSFGKVMLS (356-367)7 12 PKCLYEMLAGQAPFEGEDEDELF713 PKCLIT KHPGKRLGCGCPEGE722 PKCLYEMLAGQPPFDGE DEEELF723 PKCFLTKHPAKRLGSGPDGE771 pi +c-I LGSGGFGSVYSG (44-55)843 syn LMIH CWKKDPEERPTF861 Gs RLLLLGAGESGK (42-5 3) 862 Gs RWLRTISVILFLNK (279-293) 871 Gi　KLLLLGAGESGK(3546)872　Gi　KWFTDDTSI ILFLNK (258271) 882 Go KFFIDTSIILFLNK ( 214-227) 892 T RYFATTS IVLFLNK (253-26 6) 894 T’ KFFAATS IVLFLNK (257-270) 901 PBK LGRGVSSVvRRC (25-36)902 PBK MYTL LAGSPPFWHRKQMLML903 PBK LVSRFLVVQPQK RYTAEE911 CGK LGVGGFGRVELV (365-376)9 12 CGK MYELLTGSPPFSGPDPMKTY913 CGK L IKKLCRDNPSERLGNLK921 MLCK LGGGKFGAVC TCT (67-79)921 MLCK TYMLLSGLSPFLGDDT ETL923 MLCK FVSN LIVKEQCARMSAAQC392cm 1sk LTEIVTHGRIPYPGMTNPEVI400 MET MLK CWHPKAG MRP (Note 2) 290 v-fms LGTGAFGL VVEA (1093-1104) 3291 v-fms LWEIFSLGLN PYPGILVNSKF (1,336-1356)292 V-fms FMQ ACWALEPTRRPTF (1379-1394)” 293 V-fms LGTGAFGKVVEA (1078-1089) 295 FMS IMQACWALEPTHRPTF296 FMS LEAGVSL VRVRGRPLMMR297 FMS LYVKDPARPWNVLAQE (9 9-114) 298 FMS VPAELVRIRGEAAQIVC31Q v -abl　LGGGQYGEVYEG(367389)311　v-abl　L WEIATYGMSPYPGIDLSQVY312 v-abl LMRACW QWNPSDRPSF320 BPK CLGTGSFGRVMLV (45-5 9) 322 BPK CIYEMAAGYPPFFADQPIQIY321B PK RDNHGSFGELALM (197-209) 323 BPK RLL RNLLQVDLTKRFGNLK340 CDC28VGEGTYGVVYK A (14-25) 352 v-fps LWEAFSLGAVPYANLSNQ QTR353 ■-Attack LMQRCWEYDPRRRRSF355 c-fps NKLAELGSEEPPPALPLQ362 v-ros LMTRCWA QDPHNRPTF366 ROS IWEILTLGHQPYPAHSNLD VL367 ROS LMTQCWAQEPDQRPTF371 HIRLGQ GSFGMVYEG (990-1001) 372 HIRLWEITSLAEQ PYQGLSNEQVL373 HIRLMRMCWQFNPNMRPTF60 0 TRK LGEGAFGKVFLA (339-350) 601 TRK L WEIFTYGKQPWYQLSNTEAI602 TRK IMRGCWQR EPSNATAS661 v-kit LWELFSLGSSPYPGMPVD SKF662 v-kit IMKTCWDADPLKRPTF401 MET LWELMTRGAPPYPD ~ 7NTFDP I (Note 2) 402 MET VMLKCWHPKAGMRPSF (Note 2) 411 F O55GFNADYEASSRC(4-17)412 FO5LSPEEEEK EKRRIRKGTEYETD414 c-fos TLQAETDQLEDE KSALQTE 1415 c-fos LQTE IANLLKEKEKLE FI416 c-fos RKGSSSNEPSSDSLSSPTLL421 TGF-alpha VVSAFNDCPDSHTQFC(1-16)423 TGF-alpha FHGTCRFLyQEDKP, A(17-31)424 TGf-alpha HSGYVGVRCEHADL(34-47)431 EGF N5DSECPLSHDGYC (1-13)432 EGF CLHD GVCMYIELDKYAC442bcl-I RPPQVPAFRRPKS AEPTC461v-erb A KSFFRRTIQKNLHPTSC462 v-erb A VDFAKNLPMFSELPCEDQ463v-erbA ELPPRRCRALQILGSILPFV470 HGRKVFFKRAVE GQHNYLCAGR471 HGRNVMWLKPESTSHTLI472 HGRTNQIPKYSNGNIKKLLFHQK473 HGRVKWAKA IPGFRNLHLDDQ800 ERKAFFKR3IQGHNKYMCPA 801　ERINWAKRVPGFVDLTLHDO477　cPRKVFFK RAMEGQHNYLCAGR (note 2) 1000 Beta-Ding GF ALDTNYCFSSTEKNC oncogene directly above and sequence citations v-sis-Devour et al., Brok, Naruto, Akado, Sai, Ninisni, Edan , 3179-3182 (1982).

c-sis-Johnson et al., Nymbosia, 3:921-928 (1984).

PDGF-1 - Doolittle et al., Science, 221, 275-277 (1983 ).

PDGF-2-Dowlittle et al., Science, 221, 275-277 (1983 ).

v-myb-rashi, 0tsura, science, 216.1421-1423 (198 2).

Ha+ v-ras - Dali, Science, 217.934-937 (1982).

82).

82).

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86).

83).

v-mos-Vana Beveren et al., Nature, 289, 258-262 (1 981).

c-mos-Van Beveren et al., Cell, 27.97 (1981).

MOS-Watran et al., Brok, Natl, Out, Sainie Nisnee new-bar Guman et al., Nature, 319.226 (1986).

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8 (1980).

5RC-Parker et al., Mol. Cell, Paiol, 5,831 (1985) v-fg r-Naha O et al., Science, λdandan, 63 (1984).

Zunryo-1-Oyang et al., Mbo, J.E., 4.2905 (1985) ■-±-ki Tamura et al., Nature, Dandan Yu, 205 (1982).

v-mil-Strive et al., Nature, Dandandan, 85 (1984).

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RKS-Mark et al., Blok, Natl., Akad, Sai, Ninisny.

83, 6312 (1986).

v-tel-Stephens et al., Blok, Natl., Akad, Sai, Ninisny, 80.6229 (1983).

v-fms-Hambu et al., Brok, Natl, Akad, Sai, Ninisny.

81.85 (1984).

FMS ~ Cousens et al., Nature, 320, 277 (1986).

y-ablI-Reddy et al., Brok, Natl, Akado, Sai, USE2, 80 ．． 3623 (1983).

c-abII-Bennelik et al. Cell 44:577 (1986).

C-abl I [- Bennerik et al., supra.

c-ablm-Bennelik et al., supra.

c-ablrV-Bennelik et al., supra.

BPK C-John et al., Brok, Natl, Akad, Sai, Ninisny, 78. 848 (1981).

BPK R-Shoji et al., Biohem, 22.3702 (1983).

CDC28 - Lorincz et al., Nature, 307, 183 (1984) v-fps - Shibuya et al., Dan, 30.787 (1982).

C-Ga-Hung et al., 2141 mol, Paiol, 1dan 1,175 (1985).

v-ros-Neckmeyer et al., Nis, Vairol, 53, 879 (1985) .

HIR-Ullrich et al., Nature, 313.756 (1985).

TRK-Martin-Czanka et al., Nature, 319,743 (1 tomoe-1-cell Teng et al., Kurashiki, Dan, 603 (1986).

Akira-Semba et al., Block, Natruakadosinini, 83, 5459 (19 86).

Gs-Ito et al., Proknatl, Academia USA, 83.3776 (1 986).

G. Neito et al., supra.

Go-It-et al., supra.

PBK-Reimann et al., Biohem, 23.4185 (1984).

c-fos-Van Straten et al., Blok, Natl, Akadosai.

986).

CRR-Conniely et al., Science, 233.767 (1986).

Beta TGF Desinck et al., Nature, 316, 701 (1985).

2 The positions of a number of polypeptides are not available because the reported sequences are incomplete. Ta.

3 These polypeptides have no deletions, additions or substitutions compared to the reported sequence. contains amino acid residues.

From left to right, the homologous polypeptides encoded by the above four engineered genes are from the amino terminus to the carboxy terminus (% formula%) It can be easily described as one amino acid residue sequence shown by

Here, the amino acid residue in parentheses is a substitute for the immediately preceding amino acid residue R in the formula. Ru.

Additional polypeptides useful for inducing the production of monoclonal receptors of the invention The oncogene, its position in the oncoprotein sequence and the polypeptide amino acid residue A polypeptide whose sequence is shown in Figures 20.21 and 22. These polypeptides is a well-known family of protein kinase oncoproteins (some of whose oncogenes are mentioned above). Corresponds to the array storage area in .

■, Monoclonal receptor Although the present invention considers a large number of monoclonal receptors, a relatively small number of such receptors are considered. Only for the condition, in the form of a complete monoclonal antibody (Mab), as a representative of the group. will be described in detail in this specification. For the immunogenicity and antibody properties of polypeptides, The test was applied to different monoclonal receptors that bind (immune reactions) to different cancer proteins. The corresponding polypeptides are described below.

A. Examples of receptors Using the methods described herein, receptors can be activated for oncogene-associated polypeptides. It was.

Hybridomas secreting the monoclonal receptor of the present invention are subject to the Budapest Treaty. Hence the American Type Culture Collection of Rockville, M. Deposited in D. These ATCC accession numbers (ATCCNo.), laboratory reference numbers No. (Ref No.), data received by ATCC (ATCCRECEIPT) and Number of immunizing polypeptides cross-controlled to polypeptides in Table 1 (POLYPE P No.) are presented below in Table 2.

! blue HB8594 P43DO908102/84 125HB8595 522C O608102/84 125HB8596 510FO308102/84 125HB8679 1/24-24EO512/12/84 142HB88 00 18-9B1005109/85 112HB8888 133-IEI O08/15/85 133HB8894 173-ICII 08/27/8 5 173HB8895 202-11AB 08/27/85 202HB8 896 173-8D208/27/85 173HB8897 133-6C 1008/27/85 133HB8898 203-7D10 08/27/ 85 203HB8899 203-6F508/27/85 203HB89 00 202-9D10 08/27/85 202HB8924 132-7 C908/29/85 132HB8925 114-50D4 08/29/ 85 114HB8926 114-50G2 08/29/85 114HB 8927 132-IC808/29/85 132HB8948 121-I F912103/85 121HB8949 121-3H512103/85 121HB8950 121-4F812103/85 121HB8951 121-5ES 12/23/85 121HB8952 121-9G10 12103/85 121HB8953 121-9E5 12103/87 121HB8954 121-15B10 12103/85 121HB8 955 121-19B10 12103/85 121HB8956 121 -8D8 12104/85 121HB8965 127-24C712/1 1/85 127HB8966 127-24E11 12/11/85 12 7HB8967 127-38G2 12/11/85 127HB8968 127-50D4 12/11/85 127HB8969 127-50DI 2 12/11/85 127HB8970 127-53F8 12/11/ 85 127HB8971 127-60F3 12/11/85 12712 7-42C11127 HB8976 155-11C712/17/85 155HB8996 15 2-6D11 01/28/86 152HB8997 146-3E4 01 /28/86 146HB8998 146-17A3 01/28/86 1 46HB8999 146-8D11 01/28/86 146HB9000 155-9F6 01/28/86 155HB9001 155-8G1 01/28/86 155HB9002 310-5F5 01/28/86 310HB9003 131-94H401/28/86 131HB9004 172-12G7 01/28/86 172HB9005 172-12A 4 01/28/86 172HB9040 164-3F303/19/86 164HB9052 222-35C803/27/86 222HB905 3 310-29F7 03/27/86 310HB9077 133-10 F6 04/17/86 133HB9097 171-10ES 05108 /86 171HB9117 171-11B9 05/29/86 171H B9133 2904E1006/26/86 290HB9144 240- 13DI0 07/10/86 240HB9208 312-13EO809 /24/86 312HB9227 361-31CO510/15/86 3 16HB9260 250-9GO611106/86 250HB9278 147-67C611/20/86 147HB9279 165-34E4 11/20/86 165HB9280 360-27ED6 11/20/8 6 3601 hybridoma P44E11 is myeloma cell line P3X63 - produced using Ag8.653. All other hybridomas are were generated using the mini-Roma cell line 5P2-0 as described in the Methods section. .

Five exemplary monoclonal receptor-secreting hybridomas are listed below. fes-related 30-mar immunogenic synthetic polypeptide (also known as polypeptide a) 125), each of which also has the following carboxylic acid: terminal 12-mar polypeptide S peptide (also cited as polypeptide b) It binds to polypeptide number 126) and also binds to the v-fes gene of 5T-FeSV. It binds to the oncoprotein p85 (Dalton at 85) encoded by this The hybridoma of et al. has reference number: 510F03.522CO6, P43D09 , P42C10 and P44E11. From left to right and from the amine end Amino acid residue sequences of synthetic polypeptides (a) and (b) in the direction of the carboxy terminus is expressed by the following formula.

Polypeptide a 5DVWSFGILLIETFSLGASP-YPNLSNQ QDingR; polypeptide b 5PYPNLSNQQTR Table 2 deposited with ATCC The seven hybridomas isolated against v-fes-related polypeptide number 125 Among the 19 hybridomas generated against the polypeptide and shown in Table 1, by. The monoclonal receptors secreted by these seven hybridomas also , binds to the p85 gene protein.

The books secreted by hybridomas named 522CO6 and 510FO3 The monoclonal receptor of the invention is particularly preferably a monoclonal receptor. good together Preferred monoclonal receptors have a kappa light chain and are conjugated with the immunizing polypeptide. In addition, a fes-related protein having an amino acid residue sequence corresponding to that of the immunizing polypeptide It is an IgGI monoclonal receptor that immunoreacts with cancer proteins.

The hybridoma is a ras 23-mer immunogenic, synthetic polypeptide shown below. It was generated using code number 142 (ras).

YREQIKRVKDSDDVPMVLVGNKC monoclonal antibody is Breedomas have been shown to contain immunogenic polypeptides and Harvey sequence r. Secreted by binding to the 55-Dalton protein encoded by the as gene did. The monoclonal antibody showed that 23 in all ras-producing cell lines tested. recognizes Dalton proteins and higher molecular weight proteins.

510F03.522C06, P43DO9, P44E11 and 1/24/E The hybridoma, designated O5, has a monoclonal secretes receptors.

The five finally named hybridomas were generated by three different cell fusions. It was done. The Mabs are immunogenic polypeptides as well as their initial preparation counterparts. The production efficiency of 1 hybridoma (which recognizes cancer protein molecule ligands) is 100%. Met. That is, the two Mabs (from S10FO3 and 522CO6) are Generating those that recognize polypeptides, these two Mabs also recognize oncoproteins. I recognized it. For the second and third preparations, similarly calculated efficiencies (approximately 20 It was a percentage.

Other hybridomas include erb-B related, 16-mer immunogens shown below. was produced using synthetic polypeptide number 171. Amino acids in synthetic polypeptides Residue sequences are from left to right and from N-terminus to C-terminus in the formula Indicated by IMVKCWMIDADSRPKE.

The monoclonal antibody secreted by this hybridoma is also fes.

Regarding oncoproteins encoded by fms, alb, src and fgr oncogenes binds to the associated polypeptide.

Figure 1 shows the p85 oncoprotein ligand as an external standard and as a necatib control. hybridoma 510FO using a magglutinin-recognizing Mab. 3 (ATCCHB 8596) and 522CO6 (ATCCHB 8595) Immunological assay of p85 oncoprotein ligand by monoclonal receptor secreted by Figure 2 illustrating the production of Mab from Nozobridoma 810FO3 as well as hybridomas P43DO9 (ATCCHB 8594) and P44E11 ( ATCCHB 8593), and also Mab from hybridoma P42C10 A similar result using . gp70 low shell virus whitening Noclonal Antibodies [Nyman and Elder, Monoclonal Antibodies and T Cell Products, supra] was used as a necatib control.

FIG. 3 further illustrates the specificity of the monoclonal receptors of the invention. Therefore, C CL64 mink cells (lanes B and C) and by FeLV-B and Fe5V. Infected MSTF cells (lanes A and B) were radioactively labeled with 32p. It was. Next, extracts from the labeled cells are co-coated with the deaf portion of the V-fes gene. against the p15 protein, which is expressed as a protein precursor called pr65. Goat antiserum (lanes A and B) or tissue from hybridoma 310FO3 Culture supernatant (lanes C and D) was incubated.

As can be seen from the figure, the Mab of the present invention from hybridoma 810FO3 was p 85 protein ligand, whereas goat anti-p15 serum binds only to infected cells. (Lane A). uninfected cells Proteins from 2000 to 2000 are not bound (lanes B and D). Regarding these results and Fig. From similar evaluation studies, it was found that the Mab of the present invention has a part of the amino acid residue sequence The sequence of immunogenic polypeptides used to create hybridomas that secrete ab Confirm that it binds only to the cancer protein corresponding to the column.

In similar results, not shown, Ma b was also expressed in cells transformed by GA-FeSV. It also bound to Dalton's oncoprotein ligand. encoded by the GA-FeSV strain Oncoprotein ligands are immunogenically useful polypeptides with amino acid residue sequences. essentially the oncoprotein ligand encoded by the 5T-FeSV strain in the region of It's the same. See Hampe et al., Cell, 1982, 777-785.

None of the five Mabs mentioned above have the V-chiba gene of the Fujinami strain of avian sarcoma virus. It did not bind to the oncoprotein encoded by. Predicted v-fps oncoproteins, The sequence of Shibuya et al., Cell, 30.787 (contains strong homology to white and In the region corresponding to the above-mentioned 12-mer (polypeptide (b)), differs from the amino terminus of this 12-mar polypeptide only in the substitution of four residues. That is, the amino-terminal serine (S) of C.f.-related polypeptides and oncoproteins is , substituted by valine (V) in v-fps-related oncoproteins, from the amino terminus The second proline (P) residue is replaced with an alanine (A) residue.

I can do it. This difference in binding also explains the use of the present invention as part of the affinity adsorbent described below. Purify the mixture of two proteins by affinity chromatography using Mab. It is also useful when manufacturing.

The monoclonal antibody of the present invention directed against v-fps-related oncoprotein In addition, the uniqueness of monoclonal receptors compared to conventionally obtained oligoclonal receptors Make noticeable improvements in sex. Namely Sen, Brok, Natl, Akado, Sai, Ninisny, 80.1246-1250 (1983), Rabbit Oligochrome The above polypeptide (b) conjugated to KLH was used to generate a null antibody. . It binds to oncoproteins expressed in cells transformed by N. sarcoma virus). It matched. Therefore, the specificity obtained from the monoclonal receptors of the present invention is This is a vast improvement over that obtained with clonal receptors (both use the same immunogen potency). It can be seen that even when raised against lipeptides).

, Wang, ±, Asahi, bas, 1nt-1, for, erb-A, erb-B, mh t, EPDGFI, PDGF-2, EGF, TGF-alpha coded immunogenic polypeptides corresponding to sequences of oncoproteins and also containing these genes. A model that binds to oncoproteins expressed in cells transformed by retroviruses. Hybridomas secreting noclonal receptors are produced. Specific items of the present invention Clonal receptors act on immunogenic polypeptides encoded by the oncogenes mentioned above. Join.

Some of these oncogenes are named in Table 3 below and are preferred monochromes of the present invention. Oncogenes to which the local receptor binds, sequences and polypeptide numbers in Table 1 shown next to the polypeptide number associated with the number.

binding of at least one monoclonal receptor (Mab) or Concerning the oligoclonal antiserum (serum) produced. western plot review The data in terms of titer are also shown next to the polypeptide number in Table 3.

Table 3 Receptors that bind to cancer proteins Polypeb Binds to oncoprotein 3 Binds to oncoprotein 4 122 NT NT 123 NT + 124 NT NT 144 NT NT 145 NT + 146 + NT bas 149 + NT Sirc 151 NT + 154 NT NT 155 + + 156 NT NT 162 NT + 163 NT + Σ Engineering 201 + NT 2O2+ 10 203 + NT Average 211 NT NT 213 + NT int-’ 221 NT NT 222 + NT 292 + NT 312 + NT Engineering 360 + NT 361 + NT 416 + NT TGF-Alpha 421 10 NT 1. Binding of receptor molecules to cancer proteins in Western plot evaluation. plus title (+) indicates binding is seen. NT = not tested 2 Voripeb, No. = Polypeptide No. 3 Monoclonal Receptor from Table 1 Binding of 4 oligoclonal anti-polypeptide serum to cancer proteins useful for inducing the production of oligoclonal receptors, and eventually monoclonal Polypeptides useful in the production of receptors are preferably conjugated to a carrier as described herein. A polypeptide conjugated to a molecule, here a KLH, is an exemplary polypeptide. It has always been used as a do-carrier conjugate. Polymers containing less than about 35 amino acid residues For repeptides, induces production of oligoclonal and monoclonal receptors It is preferable to use a carrier for this purpose. Contains about 35 to about 40 amino acid residues Using the polypeptide alone, without conjugation to a carrier, to induce receptor production be able to. However, it is still preferable to use carriers for the production of these receptors. Delicious. That is, the receptor can be used for the polypeptide alone or conjugated to a carrier. Tide-induced vines or produced against these.

B. Immune binding research As mentioned several times, secretes oligoclonal antibodies and monoclonal antibodies. The polypeptides used to generate hybridomas are themselves immunogenic and anti-inflammatory. these properties make the polypeptide useful for hybridoma preparation. Provide judgment criteria for identification. The following discussion is based on rasSsis, erb- Monoclonal receptors (antibiotics) for oncoproteins encoded by B and S oncogenes. induced by the polypeptide used in the preparation of hybridomas that secrete Oligoclonal antibody (receptor)-containing antiblood produced or raised against Related to research on Qing. As described later, sis-related polypeptides , not only for this polypeptide but also for the corresponding oncoprotein, human platelet-derived growth factor. It induces the production of oligoclonal receptors that also bind to children (PDGF).

The oligoclonal antibodies produced in this way are directed against the immunizing polypeptide. 50 percent binding titer of the monoclonal antibody of the present invention. (receptors) are also produced by fusion of antibody-producing human cells with cells of the appropriate myeloma lineage. showed that it can be done.

PDGF isolated from platelets has two proteins that are approximately 60% homologous at their amino termini. It consists of a chain. One of these molecules (PDGF-2) is that the DGF-2 molecule is p28S It arises from a larger precursor with significant homology to iS. p28sis and PD The homology between GF-2 is at amino acid residue 67 of p28sis and of PDGF-2. The p It extended to the predicted carboxy terminus of 28sis. Josephs et al., Sci. p28sis is rapidly dissociated and shares the same amino terminus as PDGF-2. Generates long-lasting p20sis. encodes p20sis and PDGF-2 Within the region, there are eight amino acid changes that can be placed within three regions. Near the amino terminal Two changes are conservative, and five changes are clustered near the center of the molecule. One change is located at the carboxyl terminal portion.

Two exemplary polypeptides were created. Also cited as polypeptide (C) The first one, called polypeptide number 113, is a protein called p28sis. amino acids at residues 139-155 of the predicted sequence of the Lesarcoma virus transforming protein. Corresponding in residue sequence. Deval et al., Brok, Natl, Akado, Sai, Nini Snee, 80.731-735 (1983). The sequence of polypeptide (C) is also , the amino terminal of the protein chain called FDCF-2 of the human platelet-derived growth factor mentioned above. This corresponds to the arrangement of positions 73-89. A second white prediction called polypeptide (d) corresponds in the amino acid residue sequence to residues 2 to 18 of the given sequence. Rushlow et al. Science, 216.1421-1423 (1982). Polypeptide (c) and The amino acid residue sequences of (d) are shown from left to right and from the amino terminus to the carbo terminus. In the direction shown below: Polypeptide (c) RKIEIVRKKP I FKKA TV; Polypeptide (d) RRKVEQEGYPQESSKAG0 polypeptide Each of the compounds was synthesized and the Cys at their carboxy terminus (not shown in the above formula) residues to KLH, and each resulting conjugate was then combined in the Materials and Methods section. It was used to immunize mice as described generally. From the inspection in Figure 4, it can be determined that As shown, serum raised against polypeptide (C) contains polypeptide and K Contains an oligoclonal receptor that binds to LH and is active against polypeptide (d). The same serum contains polypeptide (d) and a compound that binds to polypeptide (d) and KLH. It contained oligoclonal receptors. All serums are used to produce them. It did not contain receptors that would cross-react and bind to the unwanted polypeptide.

Extracts from outdated human platelets are partially purified of PDGF. was used to obtain manufactured samples. As already mentioned, PDGF is approximately Two high molecular weight polyesters with the same apparent molecular weight have a Dalton apparent molecular weight of 30. Oncoproteins that can be reductively dissociated into peptides (called PDGF-1 and -2) be.

FIG. 5 shows polypeptide (C) and An oligoclonal receptor-containing antiserum raised against (d) was used. PDGF's Showing the results of Western plot analysis: generated for polypeptide (d) Antiserum is used as a negative control. As can be seen from the inspection in Figure 5, there is little association. Oligoclonal receptor generated for polypeptide (C) Containing serum binds to three protein moieties (lane 2). One of these parts is approximately The two have an apparent molecular weight of about 30 Daltons, and the two each have an apparent molecular weight of about 16 to 18 Daltons. Ru. Lane 4 also shows oligoclonal antibodies contained in the anti-sis-related polypeptide serum. binding by receptors. Oligochrome generated for the expected butide (d) nal receptors (lanes and 5).

The amino acid residue sequences of PDGF-1 and -2 are collinear with the sequence of p28SiS ( The amino acid residue sequence of the polypeptide (C) correspond to positions 67-83 and 73-89 of PDGF-1 and -2, respectively. P The amino acid residue sequence of residues 73 to 80 of DGF-2 has been determined [Dourit et al. Le et al., Science, 221.275-277 (1983)), total of these residues. are the same as the first (amino terminal) eight residues of polypeptide (c). Canada In addition, it corresponds to residues 147-155 of the p28sis oncoprotein and from PDGF. Polypeptides are sequenced [Waterfield, Nature, 30 4.35-39 (1983)), all nine residues identified to date are The same as the corresponding residue in ripeptide (c). That is, polypeptide (C) 16 of the 17 residues in human-derived FDCF and retroviral transformed The residues are the same in both p28sis derived from the cell line and the sequence The same is true for

The above results are useful for preparing hybridomas secreting the monoclonal receptor of the present invention. Immunogenicity and antigenicity of two additional polypeptides useful for linked immunization exemplify. These results also support the oligonucleotides raised against polypeptide (C). local receptors to oncoproteins PDGF1, PDGF-1, and PDGF-2. Additional synthetic polypeptides representing various regions of the two PDGF sequences that also bind Chido was created. Amino terminus of PDGF-1 and PDGF-2 and PDGF The central and carboxyl-terminal portions of -2 were synthesized and immunogen carrier keyhole lymphase was synthesized. conjugated to hemocyanin (KLH) and the 50 percent conjugated droplet described above. in mice to induce production of oligoclonal receptor-containing antiserum showing quantitative Injected.

A polypeptide representing a unique region of FDCF-2 consists of the first 18 amino acids of this sequence. Contains amino acids and is called PDGF-2 (1-188 polypeptide number 112) . where the numbers in parentheses indicate the apex of the corresponding molecule numbered from the amine end. Shows amino acid residues. The unique region of PDGF-1 is designated as polypeptide number 111. It is represented by the polypeptide PDGF-1 (1-12), which is also cited in Contains the first 12 amino acids of this sequence. Six of these 12 amino acids are PDG It is common to F-2, but only three are consecutive as mentioned above. third port Polypeptide (C) is herein referred to as polypeptide (C) Also referred to as polypeptide number 113. It was predicted that p28sis Represents amino acid residues 139-155 and its carboxylic acid residues for coupling purposes. Contains an additional cysteine at the sill terminus. This polypeptide is coupled to KLH Purified PDGF miniaturized subunits in platelet extracts when , MW31000.30000.21000, and 18000-14000 Recognizes the Dalton protein at 56 in white and SSV-infected mercapset cells. induced the production of antibodies. The fourth polypeptide, PDGF-2 (126-145 ) is also the V-exclusion sequence (of p28sis, also referred to as polypeptide 114). predicted by residues 191-210). The amino acid sequences of these polypeptides are , as shown above.

Oligoclonal receptor-containing antihematemia generated against these synthetic polypeptide conjugates PDGF was tested with these antisera to analyze the specificity of the supernatants. Purified PD GF was reduced and electrophoresed in a polyacrylamide gel. , and transferred onto nitrocellulose using the Western blotting method (6th Figure, lanes A-F).

In lanes A and B, the two antisera against PDGF-1 (1-12) Approximately 18. There was an immunoreaction with the OOO Dalton protein. Sequence analysis of purified PDGF indicates that much of the PDGF-1 chain moves to this position [Antonides et al. , Science, 220.963-965 (1983)). Reactivity of these antisera The weakness of PDGF-1 is that the amino terminus of PDGF-1 is not easily accessible for antibody binding. It suggests that.

In contrast, antiserum against the 7mi/terminus of PDGF-2 (118X:zC) , proteins migrating around 18,000 and 14,000 daltons were easily detected, and PDG Consistent with sequence analysis of F-2 (Antonides et al., supra).゛ The antiserum elicited by PDGF-2 (73-89) was identical to that seen in lane C. gives the same activity (lane DSE). In contrast, PDGF-2(126-145) antiserum had no detectable activity against purified PDGF.

The sequence of the PDGF-2 (126-145) polypeptide is c-PD at position 145. GF (Joseph et al., supra), this amino acid residue sequence change is an epitope. It is possible to be included within the site. This means that a polypeptide consists of 20 amino acids. Residue length and changes that couple the polypeptide to the KLH antibody protein unlikely since it is present only in the carboxy-terminal position used for vinegar That is, the lack of activity is not due to the generation of cancer polypeptide-specific antibodies. Why This is because this antiserum reacts with cell-derived PDGF-like molecules. refined The size of the detected PDGF in the preparation is 14,000-18,000 Daltons. Much of this material lacks the carboxy terminus of the predicted sequence of p28sis. This suggests that all or part of the PDGF antigenic site recognized by this antiserum remove.

Whether PDGF-like proteins are also synthesized in other transformed cell lines. In order to determine the Immunization was performed with oligoclonal receptor-containing antiserum. In Figure 7, SS ■ transformation The transfected NIH3T3 cells were treated with PDGF-1 (1-12X lanes A-C, F-H and -M) and PDGF-2 (73-89X lanes D, E, I, J, N and O ) was tested with an oligoclonal receptor-containing antiserum. PDGF-2 (73-89X Figure 6, lanes D and E) of the two sera against Figure 6; The serum used in lane D showed somewhat weaker activity with purified PDGF. . However, as seen in lane D of Figure 7, the protein of about 70,000 daltons Strong reactivity was observed, which was associated with the immunizing polypeptide, PDGF-2 (73-89 (lane E) was blocked by pre-incubation in lane E). , blocked by preincubation of antiserum with PDGF-1(1-12). was not checked.

Thus, the specific reactivity with these oncoproteins by the two antisera indicates that this This may be due to accidental cross-reactivity with a small region of GF, but this molecule is Contains sequences homologous to the amino terminus of PDGF-1 and the central region of PDGF-2 shows.

The amounts of p28sis and p20sis were determined in this anti-PDGF-2 (73-89) serum. was lower than the level of detection. Similar results were obtained with another antiserum, but showed that the 20,000 Dalton band was occasionally specifically detected (data (not shown).

Analysis of extracts of two other unrelated transformed cells with these antisera gave similar results. The TRD1 cell line is a spontaneously transformed Ba1b/ This is a 3T3 strain. [Bowen-Bovera, Brok, Natruacado, Sai Ninisny, 81.2396-2400 (1984)). This strain also has 7 0.000 Dalton proteins as well as the more immunologically relevant approximately 100,000 Dalton proteins. luton (Figure 7, lane G-1). A third cell line MSTF, and mink productively infected with Cinder Seiren strains of FeLV-B and Fe5V. The lung lineage (CCL64) also expresses a protein of the same size (Figure 7, lanes indicate -0).

In addition to the 70,000 dalton oncoprotein, the antibody against PDGF-1(1-12) The oligoclonal receptor-containing antiserum detected a protein of approximately 53,000 daltons ( (data not shown). These proteins are present in cells grown for a month in the absence of serum. Serum-free medium detected in the extract and conditioned by the TRD1 cell line It is not a serum contaminant because it is found throughout the body. All cell lines studied were including these two PDGF-like proteins (see Figure 11 in the brief description of the drawings). See also:

Oncogenetically non-transformed cells (normal diploid rat smooth muscle and human lung fibers) Expression of PDGF-like molecules in various cells, including blastema cells, is dependent on other processes. Indicates that it is related to transformation. All cell lines have PDGF-1(1-12) 70.000 and It contained a 53,000 Dalton protein, but cells were The size of other proteins detected by antisera directed against determinants predicted by completely homologous in terms of size and intensity (data not shown).

The nature of these differences is currently unknown.

Similarly, each of the four immunogenic polypeptides designated below as (eh) are oligonucleotides. These immunogenic polypeptides used to induce production of local receptors , as well as each of the two oncoproteins encoded by the ras oncogene. However, it is used to induce oligoclonal receptors. these four r's The sequences of the as-related polypeptides are shown from left to right and from the amino terminus to the carboxy terminus. In the direction of %formula%) combined expression Polypeptide (eh) KLmGAVGAR(S, V, G)GVGK.

It is shown by.

where each amino acid residue in parentheses corresponds to the immediately preceding amino acid residue sequence residue in the formula. It is an alternative to The oligoclonal receptor created in this way lasts for about 1 month. 50 percent dilution of more than 1:400 after two immunizations as described below between Has a binding titer. Furthermore, induced by polypeptides (e), (f) and (h) Each of the ras-related oligoclonal receptors that have been Sarcoma cells and (b) Harvey mouse sarcoma virus infected mouse 3T3 cells. was found to bind to oncoproteins present in lysed cell extracts of (data not shown).

As seen in Figure 12, the two immunogenic polypeptides shown below (k and 1) Each of these products induces immunogenic polypeptides as well as vfes. Oligoclonals that bind to each of the two oncoproteins encoded by the ST oncogene can trigger receptors. The sequences of the two V-fes related polypeptides are from left to right. to, and from the N-terminus to the C-terminus, the formula Polypeptide k LMEQCWAYEPGQRPSP (Polypeptide 127) Polypeptide l IGRGNFGEVFSG (polypeptide 121) It is shown by.

The oligoclonal receptors induced by polypeptides (k) and (1) are human T24 bladder cancer cells and the naturally transformed mouse 3T3 cell line (lanes A and It is seen binding to oncoproteins present in the supernatant from C).

Hybridoma ATCCHB 8952. created against polypeptide 121. H The monoclonal receptors secreted by B 8954 and HB 8955 are immunoreactive with one or more proteins obtained from tumors of the rectum, stomach, and uterus. It was recognized that Polypeptide 127 (hybridoma 127-42C1) A monoclonal receptor made for Responsiveness is discussed below.

As shown in Figure 13, the protein related to the ras gene is v-rasHa ( ras synthetic polypeptide corresponding to positions 1t96-118 of polypeptide 142) Monoclonal antibody made against (hybridoma ATCCHB 8679 Detected by ). The protein was detected in lane A and the immunized polypeptide This is prevented by brain kinivation with Do (Lane B). That is, the immunization port Pre-incubation with the repeptide strongly inhibited the reactive state of the protein.

1), or to ras-related polypeptides (e-h), or the following affinity adsorption Those generated against other cancer protein-related polypeptides disclosed in the Naturally occurring proteinaceous substances that are otherwise difficult to obtain in pure form, such as PDGF To provide a simple and relatively trouble-free means for making. In other words, as described below Rather than having to go through lengthy procedures to obtain pure PDGF, simply lyse the cells and Hearts were isolated and placed on an affinity adsorbent containing bound anti-polypeptide (C) receptors. Dissolve the purified protein after clearing and dissociating the reversible ligand complexes formed. can be released. Some other proteinaceous substances are non-specific on the affinity adsorbent column. Purified protein monomers that can be bound together in a purified form but are otherwise difficult to obtain in purified form. Separation is greatly improved using such adsorbents.

Antisera against the conserved sequences mentioned above can be used to detect proteins in a wide variety of transformed cell lines. Reacts with white. The antiserum was found to be more prevalent in the urine of cancer patients and pregnant women than in normal controls. Easily detects cancer gene-related proteins that are 5 to 5.5 times more concentrated. do. Distinctive patterns of expression occur in different malignancies and at different gestational periods of pregnancy. Detected during the phase.

Anti-peptide antibodies are particularly useful for detecting proteins that are immunologically related to oncogene sequences. Suitable [Wong et al., Blok, Natl, Akado.

Cy, Ninisny, 78.7412-7416 (1981)). they are arrays Therefore, special anti-peptide antibodies can be directed to highly conserved regions of proteins. , maximizing the possibility of identifying related molecules with similar effects. anti pep Immune recognition of proteins by anti-antibodies must be highly dependent on antigen conformation. anti-protein antibodies, most of which are directed against determinants specific to folded proteins. It can recognize proteins that are not detected by the body. Finally, the anti-peptide antibody Binding is relative to modulated or fragmented forms of the target antigen that may occur in body fluids or secretions. Relatively unresponsive.

In Tables 1 and 3, the synthetic peptides used to make the antibodies are listed and other Displayed with related sequences of oncogenes. An exemplary raS polypeptide 142 is Autophosphorylation by p21 encoded by v-rasHa or v-rasKi v-rasH located at amino acids 37-59 downstream from the converted threonine residue It is an a array. The sequence is identical to H-RAS, and K-RAS has one conserved amino acid. It differs depending on the amino acid change. Kaybon et al., Nature 304.507 (1983). si The sequence of PDGF used to make monoclonal antibodies is IIJl (poly Peptide 112) is located at the N-terminus of the other chain of platelet-inducing growth factor (PDGF- It is similar to the first 12 amino acids of 1). fes peptide (polypeptide 12 7) is 85 of v-fes-st (positions 927-942 of v-fes-GA), Constituting residues 744-759 of the 000 Dalton fusion protein, major tyrosine phosphorylation 79-94 amino acids downstream from the site. The polypeptide used in this study was , were chosen because they represent highly conserved parts of individual oncogene families.

Antisera against these conserved sequences can be used to detect proteins in a wide variety of transformed cell lines. reacts. Three antisera of mink protein transformed with feline sarcoma virus. The reactivity of the thread with proteins is shown in FIG. Antibodies against sis peptide are 20,000 Dalton protein and mink filament in SSV-transformed NRK cells. The sis-related protein of approximately 56 OOO daltons (p56sis) in sample 1) was detected. put out Antibodies against the sas peptide are approximately 21,000 Daltons (p21r as) and a secondary protein of approximately 30,000 Daltons in cell extracts. Detect. Antiserum against fes protein was 85,000 dalton gas-fes fused. synthetic protein (pp85-gog-fes) and 40,000 dalton protein (p4 (1fes, lane 3) is detected.

In Figure 15, the reactivity of these antisera with urine proteins from various patients is shown. . sis antiserum was 56,000, 31,000 and 2 in urine concentrate (panel 4). Detects a protein of 5,000 daltons.

Antibodies bound to all three proteins were pre-combined with the sis peptide (panel B). reaction with ras peptide (panel A). Not by incubation. The detected protein concentrate is different from the normal carrier. 50 times higher (see below). The whole urine examined contained three sis-related proteins; , with the exception of samples from patients with lymphoma, which contain a 56,000 dalton protein. There was no (lane 4).

p55sis in urine from donors with multiple myeloma and gastric cancer (panel A).

The somewhat faster mobility of lanes 1 and 2) may be due to the excess albumin in these samples. Therefore, the strain on the low molecular weight protein in lane 1 is based on the excess amount of antibody chains. Make.

In panel C, various ras-related proteins detected in urine samples are presented.

The proteins were approximately 100,000 and 55,000 Daltons and were detected (Paper Nell C, lanes 2-4). Again, the specificity of the antiserum was determined by the ras peptide (panel D ) was shown to block the activity by incubation with Breincubation with the sis peptide (panel C) did not.

The 55,000 dalton ras-related protein is the 56,000 dalton sis-related protein. (see below), exhibiting different reactivity patterns in each sample. Protein is , panel C. is not detectable in lane 1 (gastric cancer), whereas Four horizontal stripes of intensity are seen in lane 2 (38 weeks gestation).

Strongly reactive double streaks appear when urine from breast cancer patients (donors) is probed. , visualized in lane 3. The secondary horizontal stripes at approximately 35,000 Daltons are 55 In lane 4, a single 55.,000 dalton protein binds to a high concentration of proteins. 000 Dalton horizontal stripes were detected.

A protein of approximately 21,000 daltons was detected in all four lanes of panel C. these Although the mobility of the smaller protein in panel C rune 1 is a little slower, were present in similar concentrations. This altered mobility is due to electrophoresis of the ras-encoded protein. The effect of the change at amino acid residue position 12 on dynamic mobility is significant. 25, Binding detected at 000 Daltons was determined based on co-migration with antibody chains. difficult to do.

In panel E, 35,000 and 40,000 dalton fes-related proteins are shown. be done. Binding was determined by binding with the immunizing fes peptide (panel E5 lane 1). Although inhibited by reincubation, ras peptide or erbB or abl protein (panel E, lanes 2-4) and a peptide expressing a similar sequence. by incubation.

In summary, the above-mentioned 3 antisera can be used to detect 8 different proteins in urine, 3siS-related proteins ( p56 sis, p31 sis and p25 sis), 3 ras related proteins ( pi00ras, p55ras and p21ras), and 2 fes related Proteins (p40feS, p35fes) were specifically investigated.

In Figure 23, urine of 51 controls (normal; no diagnosed tumor disease) Oncogenes in 260 urine samples from patients (donors) with various malignancies The frequency of detection of related proteins is displayed. A similar frequency in 260 urine samples from pregnancy It is shown in FIG. The amount of cancer gene-related protein in urine was determined using immunoplot. Evaluated into four categories: undetectable, detectable, 5-15x increased, 15x Rise was placed in one of the larger.

Malignant types for which 10 or more samples were tested are displayed individually. The remaining types are Displayed as a union.

p21ras was detected in approximately 70% of all tumor samples. However, similar degree of elevated levels found in the urine of breast cancer patients that are found in apparently normal individuals. In contrast to ras and fes related proteins, bladder and prostate cancer patients have elevated levels of 56,000 Dalton sis-related proteins are often secreted. This protein is described above. was detected in the absence of ras- and fes-related proteins (Fig. 15, lane 1.2). , panels A-C). In addition to the 56,000 dalton sis-related protein, these patients individuals often have elevated levels of 31,000 and/or 25,000 sis-related proteins. I often had it. Further symmetrically, urine from patients with benign prostatic nodules has elevated levels of did not contain these oncogene-related proteins (Fig. 8, lane 3, panels A-C). ).

High levels of similar proteins have also been found in patients with lung and neck cancer and non-Hodgkin's lymphoma. (See Figure 23). In these latter patients, elevated 31.0-00 and/or 25.00 Qsis-related proteins, but 56.000 Dal (Figure 5, lane 4, panels A-B).

That is, three abnormal patterns were observed in urine samples from cancer patients. Breast cancer patient A subset of individuals receive elevated levels of I with p40fes and/or p35fes. ) had 55 ras. Bladder and prostate cancer patients have p55ras, p40fe In the absence of s and p35fes, increased amounts of all three sis-associated proteins are excreted. Ru. Finally, a subset of lung cancer and lymphoma patients have elevated levels of low molecular weight molecules. 2 of the sis-related proteins were excreted. As seen from Figures 15-18 and 23. As shown in Figure 1, the pattern of expression is higher than the excretion of single cancer gene-associated proteins. Correlates well with disease stage. Apparently normal individuals have elevated levels of these proteins. Almost undetectable.

The proteins described here have been shown to be effective against cancer based on the high specific reactivity of various anti-peptide antisera. Immunologically related to gene proteins. However, the eight proteins described Only two (p21 ras, p315is) express all proteins encoded by cancer genes. do.

The p21ras protein has GTP binding activity. That is, p21ras is protein is closely associated with clefts and hence the protein is easily detected in most urine samples. That's not surprising.

Similarly, elevated levels of transcription (Tran) specific for H-ras or K-ras script) has been detected in a wide variety of malignancies, as seen here. more Additionally, antisera against ras-related products also detected elevated expression in tumor tissues. I put it out. Here, the most significant elevation of this protein was seen in the malignant urine.

The p31sis protein, one of the chains of platelet-inducing growth factor (PDGF), also was detected. The PDGF-1 chain is only 18,0 when isolated from platelets. At 18,000 daltons, a comparison of the human c-sis sequence and v-sis Luton protein and starts the larger precursor protein. Indeed, partially purified platelets Analysis of the extract reveals a protein of approximately 31° OOO daltons. PDGF is FD has strong mitogenic activity and is released from platelets at sites of tissue injury. One of the physiological effects of GF appears to be wound healing. In addition, PDGF-like substances is secreted by many transformed cell lines, and secretion occurs during development in smooth muscle cells. appears to be adjusted. That is, p3sis, like p21raS, is physiologically Important, it is not surprising that it is present in urine in normal and abnormal stages.

In addition to oncogene-encoded proteins of the expected molecular size, additional proteins were identified in this study. was detected. Their presence suggests that they exist specifically in some cancers as well as during pregnancy. This is not based on spurious cross-reactivity, as there are Furthermore, antibodies and these Protein reactions were specifically blocked with appropriate synthetic immunogens. used as an immunogen These additional proteins represent conserved sequences in cancer genetic families. Express the number of these gene families. Expression of these genes is associated with neoplasia or During pregnancy, you will undergo a correlative preparation. Regardless of the super-thickness of these proteins, they The fact that they are differentially expressed during formation and gestation makes them important markers. Make it.

■0 Diagnostic system and method A diagnostic system, preferably in kit form, is yet another embodiment of the invention. This system is useful in assessing the presence of cancer proteins by forming an immune response. This system is at least one package containing a biologically active monoclonal receptor molecule of the invention; Including cage. That is, the receptor is (a) encoded by a retroviral gene; Amino acid residue sequence corresponding to part of the amino acid residue sequence of cancer protein ligand A polypeptide comprising about 7 to about 40, preferably about 10 to about 30 amino acid residues in and (b) bind to oncoproteins encoded by retroviral genes.

A predetermined amount of a monoclonal receptor molecule in an aqueous composition containing an oncoprotein ligand. When mixed with the antigen, an immune reaction occurs and the connection between the receptor, the Gantt antibody and the antigen Create a plex. Examples of aqueous compositions containing oncoproteins include cell lysates, serum, These include, but are not limited to, plasma, urine and amniotic fluid.

In addition, screening with antisera against one or more oncogene-associated translation products It is particularly valuable to be useful for That is, the evaluation method described here is Conducted on some body fluid samples obtained from donors can produce accurate information regarding malignant conditions, pregnancy status, etc.

Mixing between the receptor and the Gantt takes place in an aqueous composition. However, the receptor or link The gund can be substantially dry and water-free prior to this mixing. vinegar i.e. hybridoma supernatant, ascites fluid or a solution of receptor in buffer solution for aqueous cell extraction. Reactants from two aqueous compositions can be mixed by mixing with: receptor is coated onto the indigo of a microtiter plate, and the ligand-containing particles are can be mixed with cell extract or serum; or the ligand can be mixed with microtiter coated on a nitrocellulose sheet after transfer from a rate wall, acrylamide gel, etc. tissue sections, and hybridoma supernatants, ascites, or It can be present in a buffered solution containing mixed receptors.

The use of exemplary diagnostic systems and methods of the present invention is illustrated in the figure descriptions. Where, Cancer coated onto nitrocellulose and then mixed with receptors of the invention Protein ligands are described in connection with Figures 1.2.5-8 and 11-14, while Incubate with hybridoma supernatant to form immunological complexes The cell extracts obtained are illustrated with respect to FIG. Cancer proteins from urine samples are ．． 10 and 15-19.

Receptors are used in conjunction with "indicating groups" or "labels." The instruction group or label is , determining whether an immune reaction has occurred and an immunological complex has been formed, and in some instances are used in conjunction with receptors as a means to determine the extent of such responses. Ru.

The indicator group can be a single atom such as iodine-125 or 131, hydrogen 3 or sulfur 35 or carbon 14, or NMR active elements such as fluorine 19 or nitrogen 15. Indicating groups can also be fluorescein, rhoda Fluorescent dyes such as MinB or enzymes such as horseradish peroxidase (HR P) or glucose oxidase and the like.

The indicator group is coupled to the receptor, such as when the antibody is labeled with 1251. Can be done. The indicator group also indicates that the receptor coupled to HRP occurs in mice. Alternatively, radioactive elements such as 125I can be obtained from Staphylococcus aureus. all or one of the other molecules that react with the receptor molecule, such as the one bound to protein A. It can also be parts or atoms.

When the indicator group is an enzyme such as HRP or glucose oxidase, the immune response In order to visualize the fact that a receptor-ligand complex is formed, Further additional agents are required.

Such additional agents for HRP include hydrogen peroxide and oxidative dye precursors. An example is diaminobenzidine. Useful for glucose oxidase Additional agents include ABTS dye, glucose and HRP.

The termindicating group or label is used herein to refer to a group attached to a receptor or separately. It is used to include single atoms and molecules. These atoms or molecules used alone regardless of whether it is used in conjunction with additional agents. such an indicator or the labels are well known per se in immunochemistry, and their otherwise novel acceptance. forms part of the invention only insofar as they are used in conjunction with the body, method, and/or system. The indicator or label is preferably supplied with the receptor and packaged together or separately. It can be done. Additional agents such as hydrogen peroxide and diaminobenzidine may also be added to HRP. If an indicator group such as is used, it can be included in the system. Such substances are , as well as many indicator groups, are readily available commercially and need to be supplied with the diagnostic system. do not have. Also, some agents, such as hydrogen peroxide, constantly decompose or otherwise become harmful ( Like some radioactive elements, they are short-lived and can be better supplied by the end user. It will be done.

This diagnostic system is also sold under the trademarks Imron■ (Dynatic, Alxa (Ndria, VA) 96-well microtiter plate solid matrix can include Microtiter strips or plates should be It consists of a plastic material, preferably polyvinyl chloride or polystyrene.

Alternative solid matrices for use in the diagnostic systems and methods of the invention include Abbott Approximately 1 micron in diameter, available from To Laboratories, North Chicago, IL. Polystyrene beads of approximately 5 mm, polystyrene chews of any size The polystyrene particles of bars, steaks, and paddles are approximately 1 micron in size. , polystyrene latex can be centrifuged from latex. .

The solid matrix may also contain various substances, such as cross-linked dextran, e.g. Adex G-25, -50, -100, -200, etc. [Pharmacia Phi Agarose and crosslinkers (available from N.C. Chemicals, Piscataway, N.J.) Bridge agarose such as Sepharose-6B, CL-6B, 4B, CL46, etc. (available from Pharmacia Fine Chemicals) The disconnection also provides standards for comparing evaluation results, and seeds in dry or liquid form. various buffers (especially to wash the microtiter plate walls and to dilute the sample) (for example, for diluting labeled reactants).

A method for assessing the presence of oncoprotein ligands in a body sample from a warm-blooded animal is another aspect of the invention. Construct the surface. According to the general evaluation method, the monoclonal receptor of the present invention The sample is mixed into an aqueous composition containing the sample to be evaluated for the presence of protein ligands. Like Alternatively, the monoclonal receptor and body sample are used in predetermined amounts.

The mixture prepared in this way causes an immune reaction between the receptor and the ligand, and a period of time sufficient to form a complex (reaction product or immune reactant) , is retained. The presence of an immune complex is then determined; Indicates the presence of oncoprotein ligands in the titanium sample. The presence of the immune complex is due to the aforementioned using a label or by immunochemistry to determine the presence of an antibody-antigen complex. as determined by other well-known means.

Specific evaluation methods are also considered. Each of these specific methods involves the three steps described above. are used, but the details of these evaluation methods differ slightly from each other.

If the sample to be evaluated is in a solid phase matrix, e.g. A solid phase plate that is fixed to a twell or nitrocellulose sheet to form a solid support. The value is particularly preferred. In such cases, the mixture and monoclonal acceptor of the sample being evaluated The container forms a solid-liquid phase mixture. The solid and liquid phases are separated after the aforementioned holding period, The presence of a liquid-receptor complex is due to the presence of receptors bound to the solid support. will be determined. The relative amount of receptor bound can be determined by evaluating the provides a determination of the amount of oncoprotein ligand present in the sample being evaluated.

The receptor molecules of the invention can also be immobilized on a solid matrix, forming a solid support. . In this case, the sample to be evaluated is added to form a solid-liquid phase mixture, and the mixture is Presence of immune complexes and oncoproteins in samples maintained and evaluated as described. The current is bound by a predetermined amount of labeled ligand, e.g. an immobilized receptor molecule. determined by a mixture of polypeptides or oncoproteins. That is, receptors and oncoproteins The presence of the complex formed between the whites provides the amount of labeled ligand binding and The amount of binding is a known control amount provided in the case of free of the protein for which the sample is being evaluated. less than. The relative amount of cancer protein in the sample can be determined by using excess receptor and labeling. This can be determined by measuring the reduced binding of conjugated ligand.

Oncoprotein ligands bound by polypeptides or receptor molecules of the invention may also be It can be attached to a matrix to form a solid support antigen. of the invention in known excess amounts. The receptor molecules are mixed with the sample to be evaluated to form a solid mixture. thus The solid mixture formed contains enough water to form the immune complex reaction product. It is held for a period of time and then mixed with a solid support to form a solid-liquid phase mixture. mixture The object is that the excess unreacted receptor molecules present are immunoreacted and complexed with the solid-supported antigen. held for a sufficient period of time to form a box. Amount of complex formed is determined using the previously described procedures after separation of the solid and liquid phases. This method is , a predetermined amount of the receptor and If a solid phase ligand is used, a determination can be made regarding its relative.

■Specific evaluation Fluid body samples can be screened with antisera against cancer gene-encoded proteins of - Screening can be accomplished systematically according to the evaluation method of the present invention. - Below Screening of samples with the above antisera will determine the presence of cancer proteins in the sample being evaluated. Provide a pattern.

In breast cancer patients, p55ras and p40fes are found in bladder and prostate cancer patients. It can be seen that p56sis is increased compared to the observed p56sis (Fig. 18). and Figure 17). Also, bladder and prostate cancer patients often have a 31 Dalton or 25 Dalton demonstrated elevated levels of Dalton sis-associated proteins. In contrast, benign prostate glands Nodule donors did not demonstrate elevated levels of these proteins.

High levels of small proteins were also found in patients with lung and neck cancer and non-Hodgkin's lymphoma. (See Figure 23). In these patients, elevated 31 to Dalton and/or 25 Dalton sis-related protein was found in 56 and in the absence of Dalton (Fig. 15). , lane 4. Panels A-B).

That is, three unusual patterns were observed in urine samples from cancer patients. breast cancer patient a subset of t) elevated levels conjugated with 40fes and/or p35fes p55ras. In contrast, patients with bladder and prostate cancer have p55ras , increased amounts of all three sis-associated in the absence of p40fes and p35fes secretes protein. Finally, a subset of lung cancer and lymphoma patients have elevated levels of Only sis-related proteins of low molecular weight size are secreted. Expression as seen from drawings pattern correlates better with disease state than the secretion of high levels of single cancer gene-associated proteins. related. In apparently normal carriers, elevated levels of these proteins are rarely detected. Not done.

The discovery of cancer gene-associated proteins in urine was unexpected and previously reported by others. It has not been. This discovery provides the basis for further method aspects of the invention. Ru.

According to this method, a urine or urine concentrate sample is immunoreacted with cancer proteins, as described above. The receptor is mixed into an aqueous composition. The mixture is formed by the immune complex The immune complex is retained for a sufficient period of time and the immune complex can be assessed using the general method described above. determined in relation to the law and specific methods mentioned above.

In this way, any receptor known for immune response with cancer proteins can be used. Immediately The receptor molecule may be of polyclonal, oligoclonal or monoclonal origin. can be whole or fusion oncoproteins, or polypeptides as described herein. It can be made against Chido.

Plotting techniques, e.g. drawing Western plot techniques and when the sample is solid attached to a nitrocellulose matrix as a support, in which the liquid aqueous composition A so-called slot plot in which the receptor molecules inside are immobilized on nitrocellulose It is the preferred technique for analysis. However, other techniques, e.g. microtiter Solid phase Elsa and Radioimmunoa using plate wells as solid matrix The dipstick method is also useful.

■, intrauterine, fetal sex determination 5 site-directed monoclonal antibody probes and A single oligoclonal serum probe detects 1betaT in urine from newborns and pregnant women. Detection of cancer proteins related to GF, EGF, 1nt-1, fes, ras and myb used to do. The submission of beta-TGF-related oncoprotein ligands Commonly found in floor samples. Submission of these samples is based on fes and ras related Contains protein-linked proteins, which, as previously discussed, were elevated in urine from breast cancer patients. male and urine samples from female newborns or pregnant women contained additional oncogene proteins. two eggs White (p55ras and p4Qfes are pregnant women carrying a 16-18 week female fetus. were found to be elevated in their urine samples. Hybrids used to make antibody probes Polypeptides and synthetic polypeptides are listed in Table 4. The samples are as described below. was screened using the Western plot technique.

Table 4 Antibody 1/ Oncogene/ Growth factor hybridoma polypeptide number beta TGF (oligonal) 1000EGF 432-25GO7432 int-1222-35CO8 222-33AO5222 fes/FES 127-42C11127src 203 07D10 20 3 H-RAS/N-RAS 142-24 E 05 142cmnyc Roux C 152-06D11 152v-myb 133-10 F 06 1332/ Each urine sample was reduced, boiled and purified as described in the Materials and Methods section. Applied to acrylamide gel. After transfer to nitrocellulose, individual samples were probed with two each antiserum. The results for 25 individuals are shown in Table 5. , in the table, the relative density values were determined optically.

1/ Oncogene growth factor, hybridoma name and number of polypeptides As shown in Table 2.

2/ This polypeptide contains one of the three adjacent histidines found in the chicken sequence. is missing.

Table 5 IBA 0 0 1 0 1 0 0 1 0 1 0 1 0 0 0 2 0 0 5 0 0VAL O00000000000000000100CI N 000 0.200 101101O0110100IME 222 10 01 13201000220530STR2220000242010001 30401WOL 303 0000 25300000030520SER2 12230202203112310301CAR3230110052000 12320402TOTAL657 2623 66804443761122 5POS, 2 (total) HOL 0 0 0 0 1 1 0 0 0 1 1 1 0 0 0 1 0 1 0 0 0 SAN 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2GEA 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 GAR0000100 00101000000001PEA 000 0 1 00 0000000 0000002SUN 000 0000 00000000000001BA RO00000000000000000001BAT OO00220001 00110100002 lid ^ [000020000000000000003S IM000 2200 00002002000003BOO00900000 000000000000 As shown in Tables 1 and 2.

Table 6 Material Urine protein level I IMF 030030 2002 43 2 00GOU 020021 00 02 22 3 33ABB 111103 1401 03 0 01GOO 330013200333532VIC2200000001330NTNT” RAS 233302 0401 22 5 41PER1100432204 00242MEZ 111212 0312 13 4 41M5 0122 00 0421 00 3 31 BLA 1111 21 2202 33 1 NTNTTER2222312302133NTNTNIM2211 33 1302 43 4 NTNTSTR2100031001330NTNT total 101377 911 88214 88 11 7PO3,! (female fetus) DUQ 0000 22 0002 22 3BOO000001000102 0 11Ac 0 1 0 3 0 3 1 0 0 3 3 2 2 ESP 01 0303 1003 32 2LOR1101000000022 BEL 320202 1002 4011AI? 1200 13 0003 23fAT 020012 1002 22TOTAL 4 6 0 3 4 7 4 0 0 7 5 7PO3,! (male fetus) 1. See notes 1 and 2 of Table 5. 'NT - Not tested.

Although some oncogenes or growth factor-related proteins were easily detected, none of the samples did not contain detectable levels of EGF-related proteins. however, All samples were tested for protein reactivity with oligoclonal antisera directed against beta-TGF. It contained. Additionally, only female samples had detectable levels of P24/P23betaT. GF (in the presence or absence of Pi2beta TGF), whereas Ishin The natal urine sample had only P12beta TGF.

Sex-linked representations of oncogene-related proteins are also shown in Table 5. A subset of female samples is , easily detectable levels of P35fes, P3gfes, P4Q Contains fes and Plooras, P55ras, P35fes, P As mentioned above, 40fes and P55ras are recommended for breast cancer patients and pregnancy support. It has already been detected at elevated levels in urine from Busset.

The pattern of oncogene-associated proteins in fetal urine showed considerable heterogeneity. some eggs Although white was uniformly detected by sex-related methods, it was necessary to further characterize these proteins. For this purpose, continuous collection of enriched materials method was probed.

Also collected serially (between 16-20 weeks) from 22 pregnant mothers. Screening by Western plot analysis using the 6 antibody probes described above It was done. The results of the screening are shown in Table 6. As seen, newborn Most proteins detected in urine are also found in normal urine. most protein concentration remained constant for 16-20 weeks, and the pattern, although unusual, was found about. Differences in pattern were uniformly detected over the 16-20 week period. It was most easily recognized by comparing the proteins produced.

In addition to proteins that were differentially constant in samples from the same individual, concentrations of other proteins has changed dramatically. For example, P24/P23 beta TGF is found in most individuals. was detected in urine from In contrast, P40fes and P55ras was detected only in the urine of mothers carrying the disease. However, having a female fetus Weekly urine collections from all patients do not contain detectable levels of these proteins. Ta.

During the one-month collection period, most of the proteins displayed were approximately half of the total, regardless of the sex of the fetus. detected in patients. In contrast to these uniformly detected proteins, P40f es and P55 ras were specifically detected in patients carrying female fetuses.

Urine from 8 patients contained detectable levels of P55ras, and 7 of these 8 patients Their urine also contained detectable levels of P40fes. All materials and new The lack of detection of sex-binding proteins in urine from infant patients may be due to the short period of expression. Ku. Daily collection of 16-20 week material beds showing protein was tested less than 1 week. Served.

Short-term detection appears to be based on hormonal regulation of expression. receive insulin Initial evaluation of pregnant patients with diabetes mellitus, beyond the extension period (at least 6 weeks) We will clarify the existence of these proteins. Furthermore, the ability to detect proteins depends on the sex of the fetus. There wasn't. Similarly, collections from normal patients with younger or older male fetuses , P55 represents the presence of ras. That is, sex-binding proteins bind to external factors or proteins. It may be a material protein induced by the temporary expression of a gene.

The above results for urine samples from pregnant (expectant) mothers indicate the sex of the fetus being carried. Provides a means to predict. As mentioned earlier, a pregnant woman carrying a male fetus did not express 40fes or P55ras proteins, while harboring female fetuses. Pregnant women express one or both of these proteins during the 16-20 weeks of pregnancy. did. Some of these pregnant women carrying female fetuses do not have access to these proteins during their pregnancy. I didn't express one side.

Pregnant women harboring male fetuses were excluded during the first 16-20 weeks, as there were no false positives. P40 fes and/or P55 ras evident in urine during pregnancy Finding oncoprotein ligands is a positive test to confirm the presence of a female fetus in the uterus. , provides an irreversibility assessment. Expressed in urine samples of pregnant women for the period 16-20 weeks The absence of P40fes and/or P55ras oncoprotein ligands indicates that Approximately 50-60% (7 of 14 and 8 of 14, respectively) of pregnant women carry a male fetus. It can be predicted that

According to this method, a sample of urine from a pregnant woman at about the first 16 to 20 weeks of pregnancy is collected. provided, preferably diluted with 2-mercaptoethanol and boiled, most preferably Preferably concentrated. The sample is (i) LMEQCWAYEPGQRPSF (first Polypeptide 12) in the table and (ii) YREQIKRVKDSDDVPMYLVGNKC (polypep of Table 1) Chido142) selected from the group consisting of, from left to right and in the N-terminus to C-terminus direction, having the formula The protein is mixed with a receptor molecule that immunoreacts with the polypeptide. The resulting mixture The receptor molecules are retained long enough to immunoreact with cancer protein ligands in the urine. exemption The existence of an infectious reaction indicates that these receptor molecules and (i) the receptor molecules are (ii) said polypeptide of about 40 Daltons immunoreactive with the receptor molecule; Dalton's 5-17 percent polyac at approximately 55 to immunoreactive with polyacid(ii). The relative molecular masses in a rylamide gel are determined between oncoprotein ligands and cancer protein ligands.

Presence of a female fetus in the uterus.

In a preferred implementation, the receptor molecules used are monoclonal. most preferred For example, monoclonal receptor molecules are referenced under reference numbers 127-42C11 and 1, respectively. Secreted by a hybridoma with 42-24EO5 (H8679).

■, Affinity adsorbent Among them, the monoclonal receptor molecule of the present invention forms an active binding moiety. Adsorbents are yet another aspect of the invention.

In this embodiment, the monoclonal receptor molecules of the invention are purified by this adsorbent. attached to a solid support that is chemically inert to the oncoprotein ligand to be produced. . The term chemically inert here refers to the chemical bond between the solid support and the oncoprotein ligand. It is used to mean that no chemical reaction occurs. However, with a solid support Physical interactions between cancer proteins, such as non-specific binding, are preferably minimized. It should happen, but it can and does happen.

The solid support can be made of various materials, such as cross-linked dextrans such as Sephadex. G-25, -50, -100, -200, etc. (Pharmacia Fine Chemica) Agarose and Cross-linked agarose such as Sepharose 6BSCL6B, 4B, CL4B, etc. (available from Hari Pharmacia Fine Chemicals) or bio-gel A-0, 5M5A-1, 5MSA-50M, etc. (Bio-Rad Laboratories) , Richsend. Sulfornia (available from ), or polyacrylamide vinyl For example, Paiogel P-2, P-30, P-100, P-300, etc. (available from Iorad Laboratories). polyac Rylamide beads have the lowest propensity for nonspecific binding of these supports, but But they also typically have low porosity, which limits their binding capacity. Agarose and A cross-linked agarose material is preferred herein and is exemplary used as a solid support. Will.

Agarose supports are typically activated for binding using dicyanbromide. Ru. The activated support is then washed and receives the activated peel without drying. combined with body molecules. The support-bound receptor is then washed and used. support Unreacted reactive groups on the body can be added to amines, such as ethanolamine or trichloride, if desired. (although these reactive groups quickly disintegrate) .

Affinity adsorbents cannot be used in a loose state, such as in a beaker or flask. or it can be packed into columns. Before use, the affinity adsorbent is , to remove non-specifically bound proteins or receptors that are unstablely bound to the support. For this purpose, it is preferable to wash in the buffer or other aqueous medium used for quantitative white matter purification. Delicious.

Amino acid residues of the polypeptide to which receptors attached to affinity adsorbents bind An aqueous composition containing an oncoprotein ligand having an amino acid residue sequence corresponding to the sequence For example, a serum or cell extract is prepared and then mixed with an affinity adsorbent.

This mixture allows for reversible binding between the bound receptor and the oncoprotein ligand. Ligand combination formation - t The bound receptor-ligand complex is then exposed to uncomplexed water. a purified form of cancer separated from the rest of the sexual composition and thereby bound to an affinity adsorbent; Get protein. If the mixing is done in a beaker or in a flask, this separation can be accomplished by filtration and washing. It can be done through cleanliness. If the adsorbent is in the column, the separation is Elution of the plexed aqueous medium and preferably subsequent purification (which may be carried out by a washing step) If it is desired that the protein product be free of affinity adsorbents, this is typically done by various means. You can get it in order. In any of these procedures, reversible binding receptor The Gantt complex consists of a support-bound receptor and an oncoprotein ligand component. This ligand is then separated from the bound receptor and freed from the affinity adsorbent. Contains purified oncoprotein.

Dissociation of reversible complexes can be accomplished in a variety of ways. With a pH value of approximately 2.5 0.2M glycine hydrochloride is typically used.

Alternatively, the bound ligand may cause a reversible complex with the receptor. binding receptors by mixing with excess amounts of the immunogenic polypeptide used for They can compete and be separated from each other. Such competition may lead to possible denaturation of the ligand. Avoid separation of mixtures with affinity adsorbents. The mixture binds receptors and oncoprotein ligands A reversible binding receptor-ligand complex is formed between the receptor and the receptor.

The ligand receptor-ligand complex is then dissolved in an uncomplexed aqueous complex. cancer in a purified form separated from the remainder of the product and thereby bound to an affinity adsorbent. Get protein. If the mixing occurs in a beaker or flask, this separation may be performed by filtration or washing. It is done through purity. If the adsorbent is in the column, the separation is performed using uncomplexed water. This is preferably carried out again by elution of the neutral medium, followed by a water washing step.

When purified protein is desired free from an affinity adsorbent, it is typically purified by a variety of means. can be obtained. In any of these means, the reversible binding receptor-ligand The complex is dissolved into the component parts of the support-bound receptor and oncoprotein ligand, and then The binding receptor is separated from the binding receptor, and free purified protein is removed from the affinity adsorbent. provide.

Reversible dissociation of the complex may be effective in many ways. 0.2M glycine A pH value of about 2.5 for the hydrogen chloride solution is typically utilized. Separately, the bound ligand However, the reversible complex and the excess immunogenic polypeptide used to create the receptor Can be competed away from binding receptors by mixing with peptides. Such conflicts avoid possible deformation of the command. Separation of dissociated oncoprotein ligands from affinity adsorbents The separation is obtained as described above.

The preparation of affinity adsorbents and their use is widespread and ancient. However, the receptor molecule of the present invention Such materials and uses were not previously available. antigen is bound to the support A detailed description of the affinity adsorbent, its SIl preparation and use is provided by Antibody and As a Tool, edited by Marcaronis and Wahl, Joan Wiley & Sands, New York, pp. 64-67 and 76-96 (1982). .

■、Panel evaluation A panel of antibodies is a combination of antibody/antigen complexes formed in an unknown sample. Any biological sample can be analyzed by comparing combinations obtained from known body samples. It can also be used to characterize in nature. Tissue, urine or other body fluids are The vines have been characterized with respect to expression of offspring and oncogene-related sequences. This expression is hidden is interpreted to indicate the developmental stage of the tissue or fetus or the presence or severity of cancer.

Panel evaluation involves screening samples with one or more antibodies. Each sample complex between the antibody and the ligand found in the sample that is reacted with the antibody detected. A pattern of complex, this is Riggan among the unknown This is a combination of antibodies that react with a compound, and is compared with the pattern of reactions with antibodies in known samples. Ru. The more similar the combinations are, i.e. both known and unknown samples will have the same trigger. The higher the match of the same antibody reacting with the sample, the more similar the samples are. In other words, Known samples may be at the same stage of development or have the same disease status as known samples. Highly sexual.

Evaluation can be performed with the ligand as described above or by using a solid substrate. . Here, some biological body samples are electrophoresed into a gel and onto nitrocellulose. It is then cut into strips. This Unistern plot The trial recognizes that the same sample can be probed with a panel of antibodies. nitrocellulose The identity of specific and non-specific horizontal stripes is tolerated by recreating the base mass of the stripes. Easy to determine. For cell or tissue extracts, approximately 0.5 u of protein per gel was placed in For urine samples, an equivalent volume of 12 ml of urine was added and other body fluids (blood For serum, plasma, amniotic fluid, follicular fluid, ascites, saliva), 100 μl was placed. each In addition to probing samples with several antibodies, more than 24 samples can be probed with the same antibody. probed. This approach yields different avidity based on different antigen concentrations, No variability based on secondary reagents or incubation conditions.

Binding can also be semi-quantitated depending on the sample being probed, and the sample is serially diluted. to obtain a relative increase in concentration above the detectable level.

The use of a solid support provides a means of trial analysis using an automated scanner. These spaces Canner digitizes information onto nitrocellulose. It can be programmed to compare samples, and it can also be programmed to compare samples. this In this way, panel evaluation using antibody multiplicity can be performed automatically. This is an example For example, characterizing a large number of known samples to determine common properties and It can be used to characterize large numbers of unknown samples. This technique can also be used to record scores. of reactivity patterns such that recording is carried out through the use of digitized information. Producing accurate scoring records.

Samples can also be probed simultaneously with different antibodies by using cocktails of antibodies. Ru. This attempt provides a more effective means of creating and analyzing patterns of reactivity. It may create complex patterns that would be difficult to understand.

Here, separate strips of nitrocellulose are run, each carrying a certain amount of sample. containing lanes with and were probed separately.

In advance, a sample of known origin has a profile to which an unknown sample can be compared. probed to obtain. Various attempts have been made to induce such a profile. It was used in In one attempt, several antibodies were identified that recognized the same protein.

This approach increases the accuracy of profile identification. A second attempt was made using the same panel of antibodies. The purpose of this study was to profile different tumors. In contrast, this is an unknown sample. created various patterns that could be compared. A third attempt was developed for different organs. The goal was to profile normal tissue to develop a profile. this All attempts have been made to create a countermeasure for the known sample against which the pattern of the unknown sample can be compared. It was taken to use a pattern of responsiveness.

An example of using hybridomas with different specificities is the domain of a kinase gene. The fes antibody produces different patterns of reactivity towards ATP binding provided. The same pattern of cross-activity classifies and tests various biological analytes Antibodies are detected. In FIG. 25, the cell line containing the fes oncogene product is Two parts of the fes sequence as well as another oncogene, the group created for erbB. probed with antibodies. In lane ■, the fes gene product is can be easily detected by antibodies raised against the fes sequence located in the kinase region of Served. Overexposure of the gel to the fes gene by antibodies used in lanes A-H shows binding to the product, but the binding activity is significantly less than the antibody used in lane I. . As other controls, the two antibodies in lanes J and K, respectively, b B-related protein was recognized.

In Figure 26, the same antibody is directed against the EGF receptor (which similar to probing extracts from cell lines containing used sometimes. This protein can be used in conjunction with erbB antibodies as well as fes-transformed cells. It is easily detected by P130erbB, which is found in the cells (Fig. 25). Leh lane D, the fes antibody used for FSG was used to detect an additional protein P that was not detected in lane E. 30 fes was detected.

For ELISA evaluation, the cross-reactivity pattern to this antibody, however, was It was shown to be similar to the antibody used in lanes F and G, and the same as the antibody used in lanes F and G.

These same antibodies were used in Figure 27 to probe concentrated urine samples from gestational diabetes patients. Robed. A protein of approximately 70 Kd was detected by the three antibodies used in lanes A-C. On the other hand, a 55Kd protein was detected in lanes DH. That is, these fes proteins The white cross-reactivity pattern identifies antibodies that likely recognize the same protein. It is useful for Slight differences in reactivity patterns may be observed for some on a single protein. can be used to indicate the presence of cancer gene-associated determinants. This is 55Kd Five antibodies detect the protein and recognize at least three different fes-related epitopes. indicated by the body. P7 Qfes, P55fes and P The ability of the antibody to detect 30fes also depends on the ATP-binding domain of the fes oncogene product. Post-translational modifications may lead to differences in the main conformation.

Profiles of tumors derived from cell lines deposited at the NIT depository, e.g. Also provides a target for antibody recognition. In Figures 28-31, various tumors Extracts were probed with fes or erbB antibodies. Lanes A-D are Kinner probed with an antibody directed against the part of ATP that binds to a site in the enzyme gene. . Lanes E-H were probed with antibodies directed against different parts of the same binding site. Ta. Lanes I and J are of v-erbB (1731C11 and 173-4A11). probed with antibodies directed against the amino terminus, while lanes K and L are probed with antibodies directed against the amino terminus, while lanes K and L are probed with an antibody directed against the enzyme domain.

These blobbings require a minimal amount of material (0.5 g of tissue probed with 64 antibodies). ), creating a broad-spectrum reactivity pattern.

In Figure 28, the endometrial cancer extract contains low levels of P60fes (lane A). -D) and P2O (lanes E-H), but -10,000 P2O0erb B (lane LJ) is easily detected. P200erbB has high levels in the fetal heart. Already detected in Bell (data not shown). This expression of P220erbB is , appears to be inadequate in terms of time and organization.

In Figure 29, metastatic pore tumor extracts were probed with the same antibodies. In this tumor In this case, P70fes is easily detected in the absence of P60fes. er b Antibodies directed against the amino terminus of B are P3QerbB and P35erbB and detected P40erbB, while directed to the carboxyl moiety of the viral protein. The antibody detects P130erbB.

In Figure 30, other breast cancer extracts produced other reactivity patterns. P7 In addition to Qfes, weak activity was detected in P80fes. These proteins both, although this group recognized at least three different epitopes. It consisted of all four antibodies. erb B double stripes, P 130 erbB too, It was also weakly detected (L in lane).

In Figure 31, ovarian cancer produced a different reactivity pattern. P2Ofes is P 35 fes was weakly detected (lanes E-H) (lanes A- D), while P70fes was P35fesB (lane I , J) easily detected (lanes A-D). That is, each tumor has a unique pattern of reactivity. Each pattern noted was detected by at least two antibodies. lane The proteins in E-H contained at least three fes-related epitopes. protein is scary Although it was not encoded by the fes oncogene.

In Figures 32-39, several additional oncogene-related proteins were detected in other panels of antibodies. was detected. In lane A of each figure, the ros sequence of the conserved kinase gene is antibodies against different kinase domains were used, while in lane B Antibodies were used. In lanes C-G, 5 for the amino terminus of βTGF. antibodies were used as probes. Unique carboxyl moiety of H-ras sequence Three antibodies against the erbA hormone were used in lanes H-J, while the erbA hormone Four antibodies against conserved parts in the binding domain were used in lanes -N. Ta.

In Figure 32, breast cancer extracts were probed with this series of antibodies. Lane A In and B, the 150Kd protein has a kinase domain suggesting extensive kinase similarity. r directed to two different conserved parts.

Detected with S and fes antibodies. Ros antibody also detects 120 and 40Kd proteins did. In lanes CG, 25Kd protein was detected by total βTGF. child These antibodies recognize at least three different epitopes based on immunoplots. (See additional horizontal stripes at 45 Kd in lane C). In lanes H-J, 5 Different ras-related proteins were detected. Binding activity to these ras-related proteins The sex ratio suggests that each of these antibodies detects a unique determinant. Re Antibodies (146-3E4) in case H are P200ras, P48ras and P27ras were selectively detected. In contrast, the antibody used in lane J (146 -17A5) selectively detects P21 ras. In this explosion, this The difference between them can be determined most easily by comparing the intensities of P27ras and P21ras. easily seen. P27 is only weakly detected in lane J and P21 is detected in lane H. Gel overexposure detected all 5 ras-related proteins, although only weakly detected in was detected by all three antibodies.

The ovarian cancer probed in Figure 33 has P150 ras/fes detected. Figure 8 is similar, except that the 50KdβTGF protein appears in lane G, although it was not expressed. resemble The metastatic colon cancer probed in FIG. 34 also showed 45 and 50 Kd Similar except that βTGF protein is not detected. The ovary extract in Figure 35 is also In addition, P45ras and P52fes have relatively high concentrations compared to other ros-related proteins. The results are similar, except that no ROS-related proteins were observed.

The lymphoma extract probed in Figure 36 has a relatively high concentration of P27. Because it is unique. The breast cancer extract probed in FIG. A responsive pattern was generated. Smaller reactivity is ros, fes, and aS-related proteins. Although the easily detected P24βTGF was detected in P22erbA and and P55erbA. P22erbA is recognized by all 4 antibodies, while P55erbA was easily detected only by the antibody used in lane N. It was. Overexposure indicated binding to this protein by other 3erbA antibodies. P55 The different activities of erbA and P22erbA are due to the fact that both of these proteins Indicates that it has an rbA-related epitope. In addition, antibodies to glucocorticoids The antibody does not cross-react with homologous portions of the estrogen receptor or the estrogen receptor, and the antibody has a nuclear staining pattern. cause a turn (data not shown). That is, probed in Figures 32-37. In contrast to the extracted extracts, breast tumors have elevated levels of P22erbA. P2 2erbA protein is also found in the rectal tumor extract of FIG. P55erbA although was not detected even in overexposed gels. In Figure 39, Transplanted lung extract has a unique pattern of activity against P52fes and ras probes It had Although P48ras and P2O0ras were easily detected, Very little activity was seen against P27ras or P21ras. this The results resembled the profile of several fetal tissues (see below).

As these tissues develop, the 200 ras concentration decreases, while the P27 ras The concentration increased.

Although considerable diversity is seen in tumor samples, remarkable similarities are found in normal tissues. Served. These similarities dictate the developmental profile to distinct organs that are to be created. forgive. In Figure 40, the developmental profile of the rat striatum was created with 7 antibodies. Ta. In lane A, P21 ras and P25 ras are 18-day-old fetuses. was detected in By the 2nd, p15 Qras will be added to P21 ra5. P25ras is easily detected and present in consecutive panels. Re In case B, p52 ras was readily seen on day 18 of fetal development. gel exposure revealed P200 ras. By the 18th, P27ras is easy (P2O0ras was no longer detected in the exposed gel).

Concentrations of P27ra5 were highest by day 70. P21 ras is also It was easily detectable on day 70. Exposures that showed P21 ras occurred in all 5 times. Although it existed in points. In lane C, P150myc also It was a protein specific to mature animals that was barely detected but was easily seen by day 18.

In lane D, P120myb is a fetal-specific protein detected only in the fetal panel. It was white. In lane E, Plooint-1, P70int-1, P 45int-1, P90siS and p55sis are developmentally regulated I can't see it. In lane G, P60sis was highest in the fetal panel . That is, in contrast to the tumor extracts described in Figures 4-15, normal tissue cancer Gene-related protein profiles are much more uniform and well regulated.

That is, Figures 25 to 40 show that a known sample is applied with various resistances to create a reactive pattern. Show that it can be probed with the body. The above example is only within the same protein in different samples. Rather, it shows the cross-reactivity of different antibodies within different proteins.

A. Classification of known samples Tumor extracts or other body samples can also be characterized according to the various gene products produced. can be attached. Recently, various tumor extracts The presence and levels of various oncogene products were determined. these The data are shown in Figures 41 and 42. Tumor extracts were deposited in the NIH depository. The sample was electrolyzed on nitrocellulose as described above. Aerophoresis was performed and plotted. Encoded by oncogenes from various oncogene families Antibodies against the identified polypeptides were used to probe the samples.

Reactivity was recorded as the presence and level of oncogene product expression. In Figure 41 P52 ras was detected in all ovary extracts, but the highest levels Found in tumors labeled as low or palliative. P 5 Q src and P 4 8src was detected in ovarian tumors most frequently in the developed sector. P12 5ros and P15ras are detected in most tumors except in milk extracts, Expression was enriched in adenomas and these cancers were classified as minimal or palliative . P150r.

S expression is P120ros found in only half of breast tumors, and this protein is present in utero. Although detected in most other tumors except for membrane extracts, in contrast to Concentrated in breast tumors. Although P22erbA is widely distributed in their organization, , was not found in any of the extracts labeled as developed.

Additional separation of activities can be seen in Figure 42, which displays several kinase-related proteins. . In this table, high levels of P70fes in ovarian and endometrial extracts are , remarkable. Similarly, the restriction of P130erbB to ovary and crude oil production It may be a meaning.

That is, the tumor extract is characterized for oncogenes or oncogene-related products. be able to. Using specific antibodies, common proteins can be detected for specific tumor types. Ru. These patterns of reactivity are the patterns against which the unknown sample pattern is compared. It can be used as a clear standard.

B1 markers in body fluids Panel evaluation can also be used to monitor protein products in a single individual. This method The efficacy of treatment may be monitored non-invasively by screening, e.g. urine samples. It can be done. This approach yields a profile of activity patterns for a panel of antibodies. including screening of the original sample from the patient. As treatment progresses, The resulting sample is monitored using the same original sample as a standard for comparison. Fourth Figure 3 shows serial urine samples from a patient with gestational trophoblastic disease undergoing chemotherapy.

Asynchronous appearance of numerous oncogene-related proteins ance) is obvious. These data are used to monitor patients after treatment. It can be further correlated with clinical data regarding therapeutic efficacy.

Detection of C1 oncogene-related protein Antibodies against polypeptides encoded by oncogenes can be produced using conserved polypeptides or can recognize other proteins, including that protein. That is, a panel of antibodies sample by probing the sample with multiple antibodies, each directed against a distinct part of the sample. can be used to detect cancer gene-related proteins in Pattern of reactivity with different antibodies By determining , different cancer gene-related proteins can be identified.

p21 ras detected by H-ras specific antibody was detected by bi-reactive antibody. represents a subset of P21 ras detected by P21. H-ras specific antibody All samples containing P21 detected by antibody 142-24EO5 were Contains detected P21 but not conversely. Detected by H-ras specific antibody Other proteins that have been analyzed either lack the conserved ras portion or have at least a 142-24E5 Detected by 142-24EO5 showing changes to eliminate binding. I couldn't.

However, the concordance of binding by the three H-ras-specific antibodies indicates that the same protein This shows that all three antibodies were used for detection. P2O0ras, P48ra Similar rates of antibody binding to s and P27ras were obtained by the three antibodies. This suggests a strong structural similarity in the detected epitopes. , can be submitted using many techniques.

One approach is to digest labeled antigen and subject the digest to two-dimensional peptide genetic mapping. That's true. If larger molecules are not well recognized in solution, the concomitant It is possible to For example, samples containing different bindings of immune-related proteins may be Vine subjected to digestion and immunoplotted. If there is a precursor product relationship , the smallest size protein detected by the antibody should be the same (the added protein Control incubations without enzymes control for sample differences in endogenous propases. roll). Separately, partial digestion (to expose antigenic sites) and immunoaffinity purification and then immunoplots or peptide genetic mapping. Different samples ( Similar size proteins detected by different antibodies or the same antibody in urine and tissues) The identity of can be tested using a similar approach, or the isoelectric point can be tested using a two-dimensional It was possible to determine this by using a 3D and immunoplot detection. This attempt also Useful for identifying multiple forms migrating at similar speeds on acrylamide gels.

■ Serum screening in asymptomatic individuals: The following are those exposed to environmental carcinogens. This is a study of two different groups of workers known to be

None of the workers showed clinical symptoms of tumor disease. Both studies found that carcinogens We show that the most exposed individuals also exhibit aberrant expression of oncoproteins. first Examples include known carcinogens, including PCBs, asbestos, and smoking. One individual with exposure also showed 50-fold elevated levels of H-ras protein 21. child Eighteen months after screening, this individual developed a colorectal adenoma.

Removal of the adenoma restored the serum level of H-rasP21 to normal.

In a second example, foundry workers exposed to polycyclic aromatic hydrocarbon carcinogens An unusually high expression of the fes oncogene-related protein was observed. abnormal expression , found only in some workers in workplaces with the highest exposure to carcinogens. exposure Individuals who did not had undetectable levels of the cancer protein. These studies are aimed at improving cancer prediction. We demonstrate the usefulness of this method and monoclonal antibodies with respect to environmental carcinogens. Indicates important indicators of exposure. ) (-rasP21 index It is unexpectedly useful for predicting market conditions. The fes oncogene-related protein is a polycyclic aromatic Useful as an indicator of aromatic hydrocarbon exposure.

Example 1: Serum screening of workers exposed to PCBs polychlorinated biphenyls A group of 16 local government workers who may have been exposed to PCBs serum produced against the polypeptide encoded by the tumor gene. Screening was performed using clonal antibodies. The results shown here indicate that carcinogens Individuals with the worst histories of exposure to H-rasP also show the most aberrant expression of H-rasP21. to show that After 18 months of serum screening, this individual was diagnosed with colorectal adenosis. Ma has developed. After surgical removal of the adenoma, H-ras expression returned to normal. Ta.

Method 1. Personal background screen 16 local government workers cleaning PCBs containing sex-changing genes be evaluated for medical and occupational history, and physically and clinically examined. Ta. Approximately 600. Sex reversal gene oil containing OOOppmPCB and several months of continuous cleansing. Sweeping. All subjects were Caucasian males, age range 27-65, with an average age of 42. It was.

Workers are exposed to PCBs from other sources as well as other carcinogens, such as asbestos. have been physically tested, with particular reference to skin tests. . Laboratory tests include serum triglycerides, serum PCB levels, and liver function tests (S GOT, 5GPTSLDH1 alkaline phosphatase and bilirubin: Subjects were tested. Patients were advised to stop consuming ethanol for two weeks before the test, and not to take it orally during the test. (required). The test was conducted on a total of 16 subjects using a fixed method. described some dermal contact with PCB-containing transsexual gene oil during the course of this work; Additionally, two individuals had potential exposure to PCB-containing sex-changed gene oil over the past 20 years. (Patients 9 and 10). Seven workers had previously handled other carcinogenic materials. This was mainly due to asbestos exposure from insulation treatment, and one person also had significant occupational exposure to hydrogen chlorinated solvents and ionizing radiation (patient 1). 4). 12 workers are current or recent smokers (within the past 5 years) 4 people did not smoke at all. Two individuals reported arm, leg, and foot injuries shortly after exposure. (Patients 4 and 8), but physical examination revealed no evidence of No abnormalities closely related to PCB exposure were observed in these workers.

In all cases, liver function tests were within normal limits. In only three cases, serum was Liglycerides were high. One patient (patient 8) had very high serum triglyceride levels. reported to have been considered normal in the past prior to PCB exposure. However, his serum PCB level was very low. 2 other high trigs In individuals with lyceridemia, past status regarding triglyceride levels is unknown. and their serum PCB levels were relatively low. Even the best serum Individuals with PCB levels had normal triglyceride levels. these days Based on data, we found a correlation between relatively low serum PCB levels and serum triglycerides; It is not possible to draw any conclusions regarding the observed association with high PCB exposure.

As far as we know, overall serum PCB levels are demonstrated very little sex.

Generally, for levels below IQ ppb, such levels are It is often seen in normal controls and is therefore not seen with concern. However, this measurement Only one individual, patient 6, was considered to have truly elevated serum PCB levels. It will be done.

Patient 6, an individual shown to have highly aberrant ras expression, is also a known carcinogen. Quality exposure had the highest contingency. , Secret Letter 6 was a white man of 57 years old. blood At the time of routine screening, he showed no obvious symptoms of tumor disease. this individual has been in building maintenance for 25 years, including spraying and removing asbestos insulation. The use of various pesticides, including densin, and the cleaning of sex-changing gene oil, which contains PCBs. It was. He also smoked a pack of cigarettes a day for many years.

His physical examination was notable for only mild hypertension.

2. Serum screening Nyman et al., PNAS-USA 82 Near 924-7928 (1985), which Employing the urine immunoblotting technique of It was used to screen serum.

For the assay, subject serum is prepared and tested for polypeptides as described below. probed with a monoclonal antibody made using

For the assay, 100 μl of serum was added to 400 phosphate buffered saline, pH 7,4,2. 5 μm 2-mercaptoethanol and 475 μl sample buffer in deionized water ( 6.25% sodium dodecyl sulfate, 6.25% glycerol) and boil Place the device in a water bath for 5 minutes. The samples were then loaded onto a 5-17% polyacrylamide gel, Separated by electrophoresis and transferred to nitrocellulose. 3% bovine serum albumin and After blocking with PBS containing 0.1% Triton X-100, nitrocellulose A synthetic peptide expressing the predicted tumor gene (1:2000 dilution ascites) sequence. The cells were incubated overnight at 4°C with a monoclonal antibody raised against. Wash 3 times After cleaning, the nitrocellulose was treated with rabbit anti-vus IgF (1:500) for 60 minutes. , and incubated at room temperature. After washing three times or more, the nitrocellulose was washed with 12AI- Incubated with labeled protiin A (10'cpm/mA). Sensitize the bond Clean and visualized.

The primary antibody used was made against the protein sequence of the following tumor gene (No. 44 (A ) and (B) see figure): 5is (lane 1, hybrid 112-09B10 ( ATCCHB8800), sequence SLGSLTIAEPAMIAEC); fes( Lane 2, Hybrid 127-42C11 (ATCCHB561) and Lane 3. Hybrid 125-50DO4 (ATCCHB8968), sequence LMEQ CWAYEPGQRPSF Rune 15, Nozobrid 121-14co9 (AT cCHB9875), sequence IGRGNFGEVFSG (C)); 2, Hybrid 127-42C11 (ATCCHB 9561) and Hilane 3. Hybrid 125-50Do4 (ATCCHB8968), sequence LMEQ CWAYEPGQRPSF; Lane 15, Hybrid 121-14CO9 (A TCCHB9875), sequence IGRGNFGEVFSG (C)); β-TGF (Lane 4, Hybrid 100-30CO5 (ATCCHB9787) and 5, Nozobrid 100-34EO6 (ATCCHB9788), sequence AL DTNYCFSSTEKNC); 1nt-1 (lane 6, hybrid 222- 35CO8 (ATCCHB9052) and Lane 12, Hybrid 222-3 7F04 (ATCCHB9786), sequence LHNNEAGRTTVFS (C)) ; myb (lane 7, hybrid 133-10FO6 (ATCCHB9077 ), sequence LGEHHCTPSPPVDHG); src (lane 8, hybrid 203-07D10 (ATCCHB8898), sequence (C) GSSKSKPK DPSQRRH8); c-myc (lane 9, hybrid 155-11CO7 (ATCCHB 8976), Lane 13, Hybrid 155-08 GOI ( ATCCHB9001), and Lane 14, Hybrid 155-09FO6 ( ATCCHB9000), sequence CSTSSLYLQDLSAAASEC); mos (lane 10, hybrid 165-35FO2 (ATCCHB978 4), sequence LGSGGFGSVYKA (C)); H-ras (lane 11, Ibrid 142-24EO5 (ATCCHB8679), sequence YREQIKR VKDSDDVPMVLvGNKC and Lane 16, Hybrid 146-03 EO4 (ATCCHB8997), array YTLVREIRQHKLRKLNPP DESGPGC).

Results and discussion The results are shown in Table 7 below. The most striking results concern patient 6. disease Person 6 had the worst exposure to carcinogens of the 16 workers tested. this individual had the highest serum PCB levels and was also positive for asbestos exposure and smoking. . As can be seen in Table 7, this individual exhibited both fes and ras markers. The ras marker expression was well above the normal range, indicating abnormal expression. below In a follow-up study described by, this individual was found to have developed cancer and underwent surgery to remove the cancerous tissue. Upon removal of the target, ras expression returned to normal.

Results for other workers also showed some style of expression. Half of smokers (affected) 3-9) showed an aberrant fes oncogene with respect to binding mode, whereas non-smokers None of them showed this pattern. One smoker also had sis oncogene-related protein It showed an unusual pattern with respect to white.

But this is of unknown meaning. Example 2 below also uses polycyclic aromatic hydrocarbons. Only individuals known to have been exposed to primary carcinogens were found to have mutations in the fes oncogene-related protein. It is noteworthy that these individuals were also smokers.

Patterns for other oncogenes or oncogene-related proteins are normal except for ras. It was. Apart from patient 6, two other individuals had detectable levels of rasP21. did.

In both of these cases, the bands were relatively weak and no connection was seen to PCB exposure. It was. One patient (10) had a PCB level of 6°5 with a positive smoking layer. , and had a positive history of other carcinogen exposures (asbestos). other individuals (patients 11) had a PCB level of 0.3 but no other known exposed layers.

Patient 6 was particularly abnormal with respect to H-rasP21 expression. Lane 1 in Figure 44B 1 and 16, the band for P21 is seen to be very pronounced. normal , lanes 11 and 16 of Figure 44A, which do not show P21 expression in unexposed individuals. reference. The identity of P21 is found in the conserved portion of rasP21 (lane 11) and in H-ras. confirmed by the use of antibodies directed against the specific C-terminal portion, and if necessary For example, using antibodies or other receptors directed against conserved and protein-specific moieties. is a generally applicable method for confirming the identity of protein markers. is noteworthy. However, protein identity is not important; If anything, the important information is the relative presence or absence of proteins rather than the composition of the proteins themselves. is non-existent. namely, Patient 6, the individual with the most extreme exposure to known carcinogens. were also found to be the individuals with the most abnormal expression of H-rasP21. Ta.

These data indicate that the ras oncogene can be used to test whether an individual has been exposed to carcinogens. This indicates that it is useful as a screening marker for screening. Shown below The follow-up data show that this method and the receptors used are useful tools for cancer prediction. Show that something is true.

Follow-up study on patient 6 Almost 18 months after a serum screen showing high levels of ras protein, patient 6 had cancer. showed clinical symptoms. Patient 6 had rectal bleeding and colonoscopy revealed that the descending colon was A 2 cm tubular child denoma was revealed. This adenoma is surgically removed. Ta. Patient 6 was rescreened approximately 6 weeks after adenoma removal. This scree The results showed that the ras oncogene protein returned to its normal state. these styles is shown in FIG. Lane A is rasP21 18 months old showing clinical symptoms. Show expression. Lane B shows that 6 months after adenoma removal, no RAS band is visible. and

These data indicate that )(-rasP21 expression is associated with colon cancer proliferation and carcinogen release. Shows a correlation with dew. This abnormality precedes the development of clinical symptoms of cancer by 18 months. Detection of H-ras expression indicates the value of this marker as a prognostic tool for cancer.

What specific carcinogen or mixture of carcinogens causes colorectal cancer? is not clear. Patient 6 activated the ras gene from smoking, except for benzopyrene. There was no demonstrated exposure to known carcinogens. ras( Benzopyrene, dimethylbenzathracene, N-nitroso compounds and ionizing radiation ) and expression of ras in human colon cancer and carcinogens that activate precancerous colon polyps. On Quality, Plantaulauf and Pincus, Occupational Medicine , 2:27-38 (1987) and Subangidos and Kell, British Journal. See Journal of Cancer 49:681-688 (1984). Both of these The person shall be cited and made part of the description. Wiley et al., Seminars in Occupation. Pes- tional Medicine, 2:291-309 (1987). Asbestos is activated the ras oncogene by itself or in combination with other environmental carcinogens It is not clear whether However, (a) H-rasP21 is involved in tumor diseases. (b) expressed well in advance of the expression of clinical symptoms and is a powerful predictive tool; The method described provides an accurate and non-invasive screening, the results of which are useful in predicting the early onset of clinical symptoms; and (C) the records described herein. receptors, whether in the form of antibodies or fragments thereof or nucleic acids, directed against H-rasP21. It is clear that these methods are useful for detecting these predictive markers. Ru.

The explanation for the overexpression of P21 by patient 6 led to activation of the ras gene. Exposure to asbestos can cause chromosomal abnormalities. In this example, Although the mechanism of the pathogenesis is not clearly understood, environmental activation factors are known to be effective in humans. Therefore, other oncogenes may be selected as markers for this screen. However However, the chromosomal aberration-inducing effect of asbestos manifests itself in the activity of the ras gene. What is possible is possible. Asbestos fibers induce chromosomal abnormalities in cells in culture indicates gender. Jarland et al., Mutition Research, 169: 141-148 (1986) and Kelsey et al., British Journal O. See Bu Cancer 54:107-114 (1986). For example, m'/C The gene becomes active as a result of translocation of the proto-oncogene to the site where transcription is promoted. It was shown that the Ericran et al., PNAS-USA80:820-82 4 (1983). Also, a similar scenario is related to the activation of RAS, which causes asbestos. It can be visited. More recent research has shown that asbestos fibers are linked to oncogenes and promoters. Exogenous DNA sections containing the sequences are transferred into primate cells in culture and cell transformation is performed. -mation and showed scales. Apple, etc., PNAS-USA85 A670-7674 (1988), and this may be the mechanism of ras activation. .

Cells in culture with ras proto-oncogene binding to viral transcriptional promoter The introduction of the transfected cells increases the expression of the gene, leading to malignant transformation of the transferred cells. It is known to cause ‐mation. Chan et al., Nature, 279:4. 79-483 (1982).

That is, in this individual, long-term exposure to asbestos fibers caused his colonic epithelium to rasp. This may have resulted in increased expression of proto-oncogenes. This is in his serum revealed by an increase in the amount of proto-oncogene-encoded P21 protein. It was. Finally, proto-oncogene overrepresentation can lead to colonic overgrowth, which in this case is malignant. Clinically confirmed tubulovillous adenoma develops 18 months before progression to sexual growth. .

When the adenoma is surgically removed, the source of the ras-encoded P21 protein is removed; P21 protein was no longer detected in the patient's serum.

That is, this study included a group of individuals, all of whom were exposed to a known environmental carcinogen, PCBs. However, the results unexpectedly predicted tumor diseases resulting from asbestos exposure. We have generated markers that may be useful for

Example 2: Serum tumor gene proteins in foundry workers. iron castings in a well-defined occupational group with known and constant exposure to the carcinogen benzopyrene; factory workers, whose tumor gene-related markers have been linked to exposure to this carcinogen. was assayed to determine whether Benzopyrene (BP) and related products Cyclic aromatic hydrocarbons (PAHs) are harmful to smokers, coke oven workers, and foundry workers. has been linked to a greater risk of lung cancer in workers (Redmond et al., Animals on ・The New York Academy on Sciences, 271:12 (19 76); IARC, Polynuclea Aromatic Compounder, Monographs ・On the engineering risk o Chemicals to Humans, Volume 34, Part 3, Lion; IARC (1 984); IARC, Tobacco Smoking, Monographs on the Industry Analysis on the Carcinogenic Risk on Chemicals - Humans, Volume 38, Lion, IARC (1986). This kind of PA Hs has been shown to activate tumor genes both in vivo and in vitro. Indicated.

Ballmain and Pragnell, Nature 303 Near 2 (1983); Marcia Le et al., Nature 310:586 (1984). In addition, a detailed professional history In addition to being taken by this group, their exposure is related to BP's workplace air levels. It was well defined. There was a clear dose-related increase in peripheral leukemia in these workers. It is seen in the level of PAH-DNA adducts in the sphere, and Perala et al. Chi, 48:2288 (1988b), which is a DNA adduct produced by the 32P post-labeling method. consistent with the measurements. Phillips et al., Mutission Research 204:53. H1988): Heminke et al., Journal on Work Environment. and Health 14, 55 (1988).

Therefore, this group is a model individual for the study of oncogenic genes associated with occupational exposures. Indicates a group.

Materials and methods The workers in this study group, employed in a Finnish iron foundry, have already been described. (Heminke et al., 1988, supra: Perera et al., 1988b, supra). Simply put , foundry workers are given detailed industrial hygiene by two industrial hygienists familiar with the workplace. Groups according to level of exposure to BP based on live sampling data and work reports. It was divided into

This classification is considered representative of exposure over the past 5-10 years. Because this plan This is because most individuals tend to continue doing the same work and are generally long-term employees. Ru. Workers with 8-hour TWA exposure greater than 0.02μg/mS are in the high exposure group. workers with TWA exposure between 0.05 and 0.2 μg/m3 are classified as medium were classified into the intermediate exposure group. Peak BP levels as high as 2-3 μg/rr, 3 What happens to some individuals in the casting and hakeout areas of manufacturing facilities? was known. Clinical information, including tobacco consumption, is collected for all workers. Ta.

The unexposed control for comparison was PAH! Possible occupational diseases not related to exposure or cancer to The Institute of Occupational Health for evaluation of Refilled from referred patient. The control population had lower average cigarette smoking than foundry workers. but similar in terms of age and gender classification (Bellera et al., 19886; the above).

For the purpose of this study, repeated peripheral blood samples were collected from eight exposed workers (three in the high exposure group). and 5 in the intermediate exposure group) and 10 non-exposed controls (28 samples in total). 2 Or three blood samples were collected from the workers at different times. For all workers, The fee was prepared immediately after a month-long vacation and six weeks after returning to work. . Additional blood samples were collected for the next 12 months for two workers in the intermediate exposure group. I met. A single peripheral blood sample was collected for unexposed controls. Blood sample (30-50 mn) are collected in heparinized plastic tubes, coded and centrifuged.

Buffy coat, red blood cells and plasma were collected and stored frozen at -70°C until analysis. Ta. RAM-DNA adducts were determined for these same workers already reported. (Perera et al., 1988b, supra). Tests assayed for serum tumor genes The values of DNA adducts in a subset of samples are shown in Table 8. The missing value is Indicates that inappropriate amounts of DNA were found in some samples.

These samples were subjected to immunoblotting techniques previously described (Nyman et al., 1999). 85 supra: Plantorauf and Nyman, British Journal of ・Industrial Medicine, 45:689 (1988)) oncogenes (sis, fes).

B-TGF, 1nt-1, mybSsrc, myc, mos, and ras ) was blinded to the presence of encoded protein products. In summary, 1 00μl of serum to 400μt of phosphate buffered saline (PBS) pH 7, 5, 25t of 2-mercaptoethanol, 475 μl of sample buffer in deionized water (6 25% sodium dodecyl sulfate, 6.25% glycerol) and boiling water bath I was in the room for 5 minutes. The sample was then loaded onto a 5-17% polyacrylamide gel and electrolyzed. Separated by pneumophoresis and transferred to nitrocellulose. 3% bovine serum albumin and After blocking with PBS containing Ryo19 Triton X-100, nitrocellulose was removed. Synthetic polymers expressing predicted tumor gene sequences (ascites dilution 1:2000) were incubated overnight at 4°C. Incubate with a monoclonal antibody directed against the peptide. thorough washing After cleaning, the tumor gene protein-antibody band on the nitrocellulose was located using secondary anti-mouse I gG antibody and avidin-biotin-peroxidase complex (Vectastein; Colorimetry using Vecta Laboratories, Burlingame, California) It was decided that. Quantitation of a human body with a positive result will result in no detectable difference from normal This was achieved by diluting the sample several times. 5-fold increase in protein expression as positive Think of it as a result. The primary antibodies used were directed against the following oncogene protein sequences:

sis (hybrid 112-09B10) ATCCHB8800), sequence S LGSLTIAEPAMIAEC) fes (Hybrid 127-42C11) (, ATCCHB561) and 127-50DO4 (ATCCHB8968), Array LME QCWAYEPGQRPSF, and hybrid 121-14CO 9 (ATCCHB9875), sequence IGRGNFGEVFSG (C)) B-TG F (Hybrid 100-30CO5 (ATCCHB9787) and Hybrid Head 100-34EO6 (ATCCHB9788), array ALDTNYCFSS TEKNC) int-1 (hybrid 222-35CO8 (ATCCHB90 52) and hybrid 222-37FO4 (ATCCHB9786), sequence L HNNEAGRTTVFS(C))myl) (Hybrid 133-10FO 6 (ATCC) (B2O33), sequence LGEHHCTPSPPVDHG) src (Hybrid 203-07D10XATCCHB8898), sequence (C)G SSKSKPKDPSQRRH3) IIlyc (Hybrid 155-11CO 7 (ATCCHB8976), 155-08GO1 (ATCCHB9001), and 155-09FO6 (ATCCHB9000), array C3TSSLYLQD LSAAASEC) was (hybrid 165-35FO2 (ATCCHB9784), sequence L GSGGFGSVYKA (C)) ras (/’Ibrid 142-24EO5 (ATCCHB8679), array YREQIKRVKDSDDVPMVLVGM KC, and U hybrid 143-03EO4 (ATCCHB8997), sequence V TLVREIRQHKLRKLNPPDESGPGC) This im plottin In testing systems, these antibodies have been shown to give specific, sensitive and reproducible results. It was. The specificity of the antibody is determined by brain incubation with a specific peptide against the tumor gene. inhibition of activity by peptides and loss of inhibition of activity by peptides of other oncogenes. indicated by defeat (Nyman et al., 1985). Assay systems are in the nanogram range The protein can be detected using the It turns out that results can be obtained.

Results and discussion As shown in Table 8, the results showed that two different workers (patients 2 and 7) (who (known to have high (2) or intermediate (7) exposure to PAHs) were positive for increased levels of oncogene-related protein products. The test result is positive. Serum samples from each of these individuals were taken after vacation and 6 weeks after work.

There were no positive bands for the proteins of the seven oncogenes in any of the samples (sis ,　B-T　G　F　.

1nt-1, myb, src, myc, mos). This is a sample from patient 5. The injury was found to be positive for the ras oncogene-related protein product. But long In this case, two other samples from the same worker taken at different times were negative. Met. All results for serum tumor gene proteins in unexposed controls were negative. Met.

In this study, only positive results for high serum levels of tumor gene-related proteins It is noteworthy that the results were obtained for individuals in the medium and high exposure groups. Further shown in Table 8, As previously reported (Perera et al., 19886, supra), these individuals also have higher levels of PAH-DNA adducts compared to exposed individuals.

The two individuals with positive tumor gene proteins also smoked approximately one pack of cigarettes per day. Although smokers (patients 2 and 5), the main contribution of PAHs to their body burden was This appears to be due to exposure at work.

For example, an exposure of 0.2 ug/m3 means consuming 5 to 7 packs of cigarettes per day. Approximately equal to the BP dose from smoking.

Positive results for specific tumor gene expression in these workers It is known that workers are at high risk for the development of lung cancer (IARC, 1 984, above), so it is interesting. PAHs are active on the ras gene It is known that (Beaumain and Pragnell, 1983, supra, Marshall et al., Nature 310:586 (1984), also found that ras gene activity Cancer, especially non-small-cell variety (Slamon, Science 224:256 (1984); - Tony Hughes, Niney England Journal of Medicine 31 7:929 (1987); Krzrock, Cancer Research 46:153 0 (1986), workers with isolated positive results for ras protein are worthy of attention. do.

For example, in a recent study of lung cancer (Lawton Hughes, 1987, supra), 50 % of adenocarcinoma patients (5 in 10) have one type of the ras gene (k-r as), and this oncogene activity contributes to the relatively early development of lung cancer. incident and noticed that it was related to cigarette smoking. Non-small cell lung cancer patients In our own study of serum tumor gene protein levels in patients, 15 of 18 Patients had increased expression of the ras gene product, and 12 of these It turned out that he was a smoker. -Ras oncoprotein products in lung cancer patients It also showed enhanced urine levels (Nineman, 1985, supra). 16 people already Serum of workers who are no longer clinically at risk of exposure to known carcinogens. In our screening, one individual had a particularly abnormal ras gene protein. There was an expression of increase (see above). This individual also suffers from asbestos and tobacco smoking. had a bad history of exposure to carcinogens. That is, ras gene activation is particularly plays a significant role in lung carcinogenesis caused by exposure to carcinogens such as PAHs There is a possibility that However, as mentioned above, recent studies have shown that high serum RA An individual with s protein (patient 5) was positive in only one of the three samples. I found out something. information regarding this individual's risk for developing lung or other cancers. The meaning of the isolated results is also not clear.

However, as noted, two workers (patients 2 and 7) had fe tumors. There were consistently high serum levels of gene-related protein products. of these workers One person was a Zimmerman worker with high exposure to PAHs and cigarette smoking for the past 15 years. He was a caster in the manufacturing industry, and both his father and mother died of lung cancer. did. Another worker was Core with intermediate exposure to PAHs for the past nine years. It was 5 etter. Both of these also have occupational dermatitis and local skin irritation. He underwent long-term treatment with terrorism. Local exposure to steroids can cause PAHs. Did it affect metabolic activity or did dermatitis enhance skin absorption of carcinogens? It's not clear. Like ras, fes is often expressed in human lung cancer. It turns out. In one study, all four lung cancers studied had an increase in the fes gene. The test result was positive for the expression (Sulaimon, 1984, supra). blood in lung cancer patients In our previous study of tumor tumor gene proteins, 11 of 18 individuals were born with feS. Eight of these individuals were found to be cigarette smokers. Plateau levels of the fes oncogene product were also found in lung cancer patients (Nijman et al. 1985 above). Additionally, in our study of workers with clinically reduced health risks, , only heavy cigarette smokers (6 out of 12) had the feS gene in their serum. It was found to be positive for the offspring product. Furthermore, during mammalian development, fes The protein products of genes are produced in a very limited tissue-specific fashion ( Piementil, Onco Jeans, Boga Layton, CRC Bracelet (1986)) For example, in young chicken chicks between 6 and 18 days old, f The es gene product is detected at high levels only in three tissues - bone marrow, liver and lungs. (Maseivrevoto et al., Cell 28:897 (1982)). In other words, lung cancer , expression of the fes gene product signals regression to early stages of lung cell development. Again, this expression refers to the exposure to carcinogens in cigarette smoking, such as PAHs. A vine especially associated with dew. Therefore, the next logical step in this study is to Workplace exposure to PAHs with high levels of fever and known high levels of PAHs H-DNA adducts and consistently high levels of fes in their serum. Two workers with oncogenic protein products were linked to the development of malignant disease, particularly cancer of the lungs. The objective is to determine which individuals in this group are most at risk.

Longer-term follow-up of this and other similar groups may reveal markers for cancer development. may be necessary before the precursor values of specific tumor gene proteins can be accurately ascertained. . However, recent results suggest that serum oncogene proteins may be associated with the development of occupational cancers. It is a useful molecular epidemiological marker for population monitoring of all risks.

Examples presented herein include monoclonal receptors, other receptors such as this Here, we investigated and used nucleic acids corresponding to oncogene-related proteins and related polypeptides. Although uses of the acid sequences are specified, methods of such production and use are within the skill of the art. within range. In addition, other body samples such as tissue or urine range from those described herein. The use of the above assays and methods is within the skill in the art.

■Materials and methods A. Growth of viruses and cell lines Uninfected mink lung cell line (CCL64), feline sarcoma virus sudanesei Productive in strain Len (ST-FeSV) and feline leukemia virus B (FeLV-B) The same strain, called MSTF, was transformed into Gardner-Als 64F infected nonproductively with feline sarcoma virus (GA-FeSV) The same strain, called 3C17, was developed by Sen et al. Cultures were performed as described in Sunny, 80.1246-1250 (1983).

Nonproductive avian myeloblastoid cell line nonproductively infected with avian myeloblastosis virus Doisberg et al., Blok, Natl, Akado, Sai, Ninisny, Eyu, 51 20-5124 (1980).

NPV/SiS and NP nonproductively infected with simian sarcoma virus (SSV) A nonproductive marmoset cell line called VI/5iSV was developed by Debare et al. , Natl, Akado, Sai, Ninisny, 80.731-735 (1983). Cultured as described. Birds infected with Fujinami sarcoma virus (FSV) The fibroblast non-productive transformed cell line was produced by Bee, Sefton of the Salk I. It was a gift from the Institute, Jola, California. non-infected ma murine NIH3T3 fibroblasts and productively infected with Harvey mouse sarcoma virus. Mouse NIH3T3 fibroblasts were cultured as described by Todaro et al. human T24 Bladder sarcoma cells by Bubenitsuk et al., India, J. Cancer, 11.765 -773 (1973).

B. Synthesis of peptides Polypeptides were synthesized using Marglin and Merrifield, A., Lev, and B. , confirmed by amino acid analysis. Sequence information is the amino acid sequence of the viral protein or derived from predictions based on nucleotide sequence ratios. The sequence information source is This was displayed in a footnote regarding cancer genes.

For polypeptides with fewer than 35 residues used in the immunization inoculum, the cis tin residue is present in the polypeptide when the corresponding oncoprotein sequence does not contain such a residue. However, it is added to the amino or carbosyl terminus of each polypeptide. Cy The s residue was used to aid in coupling to the protein carrier as described below.

When making useful synthetic polypeptides using the solid-phase method described above, amino acid residues are It was bonded to a crosslinked resin (solid phase) via an ester bond from the terminal residue. polype When the peptide is attached to the carrier via a Cys residue, this Cys residue is attached to the resin. It was conveniently used as the carboxy-terminal residue that was ester-linked.

The alpha amino group of each amino acid added typically Protection with quaternary butoxycarbonyl (t-BOC) group before addition of amino acids to the chain It was done. The t-B OC group then attaches the next amino acid to the growing polypeptide chain. Removed by standard techniques prior to addition.

Reactive amino acid side chains were also protected during polypeptide synthesis. normal side chain Protecting groups were used for the remaining amino acid residues such as: For 0-(p-bromobenzyloxycarbonyl), threonine, serine, a O-pencil for spapartic acid and glutamic acid: S- for cysteine Metquin benzyl, dinitrophenyl for histidine: 2- for lysine Chlorbenzoquine carbonyl, and tosyl for arginine.

The protected amino acid is recrystallized from a suitable solvent and subjected to thin layer chromatography. Gave more single spot. Coupling typically involves an initial N-terminal amino acid. A 100-fold molar excess of the protected amino acid and dicyclo based on the number of milliequivalents of the acid. It was carried out using both hequinlecarboimide. 2-fold molar excess of both reactants can also be used. In the case of asparaquine, equimolar amounts of N-hydroxy-benzotri The azole is added to the protected amino acid and dimethylformamide is used as the solvent. used. All coupling reactions are lysine, anal, chem, acta, 58 ．． 248-249 (1972), the picric acid test completed over 99%. Ta.

After preparation of the desired polypeptide, a portion (approximately 1 g) of the obtained protected polypeptide was ml of anisole and about 20 ml of anhydrous hydrogen fluoride at dry ice temperature. It was condensed in the reaction vessel. The resulting mixture was stirred at about 4°C for about 1 hour to dissociate the protecting group. The polypeptide was removed from the resin. After vaporizing hydrogen fluoride at a temperature of 4 °C with N2 flow , extract the residue three times with anhydrous diethyl ether to remove anisole, and remove the residue under reduced pressure. It was dried.

The dried product was extracted with a 5% acetic acid aqueous solution (3 times at 50 mA) to extract the free polypeptide. The titanate was separated from the resin. Freeze-dry the extract-containing solution to synthesize unoxidized A polypeptide was obtained.

Coupling of synthetic polypeptides to C0 carrier proteins, 477 and 478 (198 2) Maleimidobenzoyl-N-hydro as a coupling agent as described Cytin residues (Cys:C ) to the carrier protein keyhole limpet hemocyanin (KLH). Googled. If the Cys residue is the terminal residue in the sequence, additional Cys No residue was added.

Briefly, the general procedure for each polypeptide is to 4 mg of KLH in 0.25 mA of sodium buffer (pH 7.2) was added to dimethyl The obtained The mixture was stirred at room temperature for 30 minutes. At a DMF concentration where KLH is about 30% or more Since the MBS solution is insoluble, to ensure that the local concentration of DMF is not too high. Added drop by drop. The reaction product KLH-MB was treated with 5QmM sodium phosphate. Sephadex G-25 (Pharmacia) equilibrated with um buffer (pH 6,0) Chromatographica manufactured by Fine Chemicals, Biscataway, NJ) ram to remove free MBS. Column elution monitored at 280 stops KLH recovery from the product peak fractions was estimated to be approximately 80%.

The KLH-MB so made was then treated with a 5-day polyester solution dissolved in 11 buffers. reacted with peptide. The pH value of the obtained reaction composition was adjusted to 7 to 7.5, and the reaction composition was The reaction composition was stirred at room temperature for 3 hours.

D. Immunization and fusion Coupled to KLH, a polypeptide corresponding to , Monoclonal Antibodies & Ti Cell Products, Cut (Poca, Radon, Florida, CRC Press, 1982), pp21-5 1, was used to immunize 129GIX mice as described in 1. this Human liver cells from immunized mice were treated with polyethylene glycol (PEG) 150 0 was used to fuse with SP210-Ag14 myeloma cells (J.T., BA. Kerr Chemco, Filth Barb, Niney Jersey); PEG solution for fusion were made at least one month prior to use to increase fusion efficiency. SP210 -Ag14 cells do not make their own 1g molecules, thereby allowing isotype analysis and Facilitating subsequent purification, such cells also do not produce retroviruses. fusion The cells were then treated with 10% fetal bovine serum, 1.0 x 1.0-'M pipoxanthin. , lXl0-'M methotrextate, and Durbetzko's High Glucose Trace Essential Medium containing 1.6 x 10”M Thymidine ( 400m of Flow Laboratories, Inc. (Inglewood, California) resuspended in r. Next, the cells were analyzed by Nyman et al. in 30 μl plates as described in Ninisny, 1982 (supra); Made it grow.

2. sis and myb related polypeptides whose amino acid residues are protein 1) 28 Positions 139-155 of the predicted sequence of the simian sarcoma virus transforming sis and a polypeptide corresponding to residues 2-18 of the predicted sequence of avian myeloblastosis virus. Peptides (C) and (d) were synthesized and coupled to the KLH carrier as described above. I nged. The conjugate thus prepared was administered once per 129GIX mouse. The polypeptide was administered in an amount of approximately 50 μg.

On day O (zero), each conjugate was mixed with complete Freund's adjuvant and injected intraperitoneally. did. On the 19th day, each zygote was added to alum at a concentration of 519 per mA of alum. It was mixed with. Enhancement of the effect of polypeptide (C) in phosphate buffered saline Note The injection was administered intravenously on day 62. Serum containing oligoclonal antibodies is The specimen was taken by eye-level puncture on the 67th day. The second of polypeptide (d) on day 41 After alum-containing immunization, the effect of polypeptide (d) was similarly enhanced on day 143. 148 to similarly produce oligoclonal antibodies. That's it The serum obtained in this manner was tested for antigenicity of its receptors as described for Figure 4. I tested it.

In a similar manner, a polypeptide, e.g. the amino acid residue sequence shown below, can be Synthesized.

abl LMRACWQWNPSDRPSFfms EMQACWALEPTR RPTFsrc LMCQCWRKDPEERPTFLGQGCFGEVWMG GSSKSKPKDPSQRRRS fgr AMEQTWRLDPEERPTE immunity is sis and myb amino acids This was carried out in a manner similar to that described for residues.

3. ras and erb B-related polypeptide Kirsten murine sarcoma virus r Residues 96-118 of the ras polypeptide from the predicted sequence of the as oncogene and Residues 366-3 of the erbB polypeptide from chicken erythroblastoid sarcoma virus The amino acid residue sequence corresponding to KLH 81 was synthesized and described above. Coupled with carrier. The conjugate thus prepared was 129GIX Approximately 50 μg of polypeptide was administered per injection per mouse.

On day 0, each conjugate was mixed with complete Freund's adjuvant and injected intravenously. did. On day 5, serum containing oligoclonal antibodies was taken by orbital puncture. like this The serum obtained was tested for its receptor antigenicity as described in Figure 4. It will be done.

E. Antibody binding evaluation Hybridomas producing anti-polypeptide antibodies are described in the explanation of FIG. 4 and by Nyman et al. Monoclonal Antibodies and Ti Cell Products (mentioned above) Detection was performed using the enzyme-linked immunosorbent assay (ELSIA) method as described in . simply say For example, approximately 50 μmol of polypeptide is dried on a microtitration plate and The cells were fixed with a microtube and incubated with hybridoma tissue culture supernatant. completely washed After testing, hybridoma antibody binding was determined using a rabbit anti-mouse kappa chain antibody (synthon ・Pionetics, Inc., Kensington, Maryland) and then gluco Detection was performed using a goat-anti-rabbit antiserum conjugated with 1-seed oxidase. Joining is Nyman et al., Monoclonal Antibodies and T Cell Products (formerly 2.2 in the presence of glucose and horseradish peroxidase as described ゛-Azinodi[3-ethyl-benzothiazoline-sulfonate (6)) (AB TS) dye (Boehringer-Mannheim, Indianapolis, Indiana). was visualized. The isotype is derived from various rabbit anti-mouse strains, as described above. was determined by replacing waste or heavy chain serum with anti-mouse kappa chain.

F, Electrophoretic migration and immunological detection on nitrocellulose. Described in Iman et al., Virology, 123.187-205 (1982). Cell extracts were subjected to polyacrylamide gel electrophoresis, and proteins were analyzed using nitrocellulose gel. (Noyureicher & Nyuell, Inc. 9. Keene, New Hampshire) Moved to

Peroxidase-labeled rabbit anti-mouse IgG blood diluted 1/1000 Sei (Dago, Inc. 1. Burlingham, California), Nyman and Elder , Monoclonal Antibodies and Ti Cell Products (cited above) ), incubate with the transfer for 1 hour at 25°C, followed by washing. It was. Bound antibodies were dissolved in 10 mM Tris(2-amino-2-(hydroxymethylene) -1,3-propanediol), pH 7.4, 0,009% H2O2, area 0 025% 3.3'-dimethquine benzidine dihydrochloride (Eastman - Incubation at Kodak, Co., Rochester, New York) More visible.

Preparation of C1 purified PDGF The 16 units of outdated platelets are Retrieved from San Diego, California. The purified PDGF used here was , Antonides et al., Blok, Natl., Akadosai.

The first two of the procedures described in Ninisny, 76.1809-1813 (1979) obtained according to the steps.

Briefly, platelets were centrifuged at 28,000 gravity (G) for 20 min at 4°C. Platelets collected by (a) 0.15 M NaCA and 1% glucose (b) 100 mj per 0.8g citric acid-hydrate, 2.2g anhydrous dextrose and 2.6g Resuspend in 4oomz of a mixture containing 1 volume of sodium citrate trihydrate of The cells were washed by washing and further centrifuged at 28,0 OOG for 10 minutes at 4°C. like this The washed platelets were then treated with 0.008M NaCl and 0.008M NaCl. OLM phosphate i 16 ml of an aqueous solution (Na(J'-phosphate ion solution) containing Na(J'-phosphate ion solution) at pH 7.4 and boiled for 10 minutes to lyse the cells.

Phenylmethyl sulfonyl fluoride and Traciylol Sigma Chemical Co., St. Louis, Sisley) and protease inhibitors at 1mM and 3%, respectively. was added to the disassembled cells at a concentration of Centrifuge the lysed cell mixture again to remove the pellets. Lett and supernatant were obtained.

The supernatant was transferred to CM Sephadex pre-equilibrated in NaCl-phosphate ion solution. C-50 (Pharmacia Fine Chemicals, Viscataway, Newge) It was mixed with 8rr+1 of Chassis) beads. NaC7-phosphate ion solution mentioned above A chromatography column (15X 1.5 cm) washed with 6 times the column volume of liquid. ) into which beads and liquid were poured. Color with twice the column volume of IM NaCl. PDGF (first eluate) was obtained by eluting the tridylol. Add the eluate to a final concentration of 3% and add the eluate to the NaCl-phosphate ion described above. Dialyzed against solution.

The lysed cell pellet made above was extracted with NaCl solution for 24 hours at 4°C. and centrifuged. The supernatant was filtered against the NaCl-phosphate ion solution described above. The mixture was analyzed and mixed with the Sephadex described above to prepare a column. Wash this column as described above. to obtain a second eluate which was dialyzed as described above. Create using this procedure. The dialyzed eluate from the three pellets was combined and filtered into an Amicon ultrafiltration device (Amicon ultrafiltration device). (Lexington, Massachusetts) and 10 to exclude Dalton. was used to concentrate to a few ml volume. The PDGF thus purified was then purified as shown in Figure 5. Processed as described above.

Approximately 2.5 units of purified PDGF extract from platelets was added to 0.5% sodium dodecyl sulfate. (SDS) and 5% 2-mercaptoethanol. combined. The resulting mixture was boiled for 2 minutes, then 5-17% polyacrylic Electrophoresis was performed through midgel. The proteins are then electrophoresed onto nitrocellulose. Moved. (Nyman and Elder, supra) It is then cut into strips and was subjected to a Western plot procedure.

The nitrocellulose strips thus prepared were then washed in phosphate buffered saline. , 3% bovine serum albumin (BSA), 0.1% polyoxyethylene-9-oct. treated with a solution containing tylphenyl ether (Triton(TM) Blocked specific protein binding.

4mf mouse antiserum diluted 1:200 and then with nitrocellulose strips. Incubated.

After washing three times with a solution of 01% Triton(TM) X-100 in PBS, the nitrocellulose 125 marked Staphylocotcus with 106 counts per minute. aureus protein or peroxidase-conjugated goat anti-mouse serum (Talo) 1.10 0.00 dilution and again with 0.1% Triton(TM) in PBS. ) Washed with X100. The peroxidase conjugate has a pH value of 7.4. 0m1! 0.0009% H2O2, 0,0025% 3,3"-di In a solution containing methoxybenzidine and hydrogen dichloride (Eastman-Kodak, Co., Ltd.) developed. 1251-labeled strip was cloned at -70°C for 48 hours. Intensifying screen By exposure on XRP-1 film (Eastman-Kodak Co., Ltd.) using developed.

H8 urine evaluation Collect urine from the donor (patient) as described in the figure legend, and either leave it collected or The solution was concentrated 40 times using an ultrafiltration device. Add this liquid to sis, fes and about proteins encoded by or related to the SaS oncogene. It was used as a fixed amount of body fluid sample in the evaluation.

Concentrated urine samples were prepared as follows. Incubate urine at 4°C for 10 minutes at 6000 rpm. It became clear. The supernatant was then filtered through an Amicon filter with a 10,000 Dalton exclusion. It was concentrated using This concentrated urine was then dialyzed to separate the protein fraction.

The concentrated bed was electrophoresed on a 5-17% polyacrylamide gel at 25 μl per run. to provide equal amounts of protein from collected urine L++1 and then electrophoresed onto nitrocellulose. It moved. Next, add a nitrocellulose filter containing, for example, 3% bovine serum albumin. 0.1% Triton(TM) X-100 and 1/2 mouse antiserum in PBS. 00 dilution was used. Next, wash the nitrocellulose filter three times. , and incubated with 1251-labeled protein A at IQ'cpm.

Bonding can be done using an intensifying screen at -70°C as described above in Figure 6. Visualized.

■, Cancer proteins and transformed cells NRK and SS■ transformed NHK cells were obtained from the Center for Cancer Lisa Varnish, National Institutes of Health, Bathesda, MD - Provided by Nie Aaronlan and K.C. Loving. The cell is 1 0% fetal bovine serum, 2mM glutamine, 100 IU/ml penicillium Durbetzko's micro-dose supplemented with 100μ9/ml streptomycin grown in a medium.

Parallel cultures of NRK and SSV-transformed NRK were washed three times at 2-hour intervals, then Incubate for 18 hours in 15++ l/T75c*2 flasks without serum in medium. I started. The conditioned medium was centrifuged and stored frozen at -70°C. I stored it.

The conditioned media was thawed, dialyzed in 1M acetic acid, concentrated 500-fold, and then Freeze and vacuum dry. Solubilization and reduction in 10% 2-mercaptoethanol Afterward, 50 μl of the concentrated conditioned medium was diluted with 5-17% sodium dodecyl sulfate. electrophoresis into a polyacrylamide gel. The secreted proteins are then subjected to electrophoresis. Dynamically transferred and bound to nitrocellulose. Non-specific binding occurs at pH 7.4. 3% bovine serum albumin and 0.1% polyoxyene in phosphate buffered saline. Brain-incubate the cell extract with a solution containing tyrene, octyl, and phenyl. I was blocked.

PDGF-2(1-18) or PDGF-2(73 -89) (described above) was added to the appropriate polypeptide. The cells were incubated at 37° C. for 1 hour with 100 μ2 of 100 μ2. oligoclonal antibodies The containing/polypeptide reaction mixture was then incubated with the above-mentioned brain incubation solution. :500 dilution. The diluted solution so prepared was then placed in a nitrocell at 4°C. contact with the loin-bound conditioned medium and maintain this contact for 15 minutes ( Incubate). This time allows the antibody (receptor) to bind to the nitrocellulose. The washed nitrocellulose is then incubated for a sufficient time for immunoreaction of the combined proteins. Affinity purified rabbit anti-mouse IgG diluted 1:500 with antibody (Litton) Contact was made at 25°C. Contact was made with anti-mouse IgG, antibody, and Antibodies from antiserum bound to secreted proteins bound to nitrocellulose in the medium It was maintained for two hours, which is sufficient for an infectious reaction. The nitrocellulose is then washed again. It was.

+5I-labeled Staphylococcus aureusp as described in 83 (1984). Visualized at 106 counts/min of Lotin A.

J Monoclonal receptors used for cancer proteins in urine samples of newborns and pregnant women have been previously Prepared as described. 1 mA of urine from each neonate was combined with enough 2-mercaptochloride. It was mixed with tanol to form a 10% by volume solution. The resulting solution was boiled for 2 minutes. cold Above all, aliquots of the resulting reduced solution were run on a 5-17% polyacrylamide gel. was electrophoresed. The proteins in the resulting gel are transferred to nitrocellulose and processed using standard procedures. Attached to the tier. The nitrocellulose plot for each urine sample is The lobes were individually screened for immunoreactivity, and such Western blots were Subjected to standard measures. Autoradiography was performed using a clone at -70°C for 4 hours. An intensifying screen was used. The relative strength of the immune response was subsequently determined.

Urine samples from pregnant (expectant) mothers were concentrated before electrophoresis. Here, pregnancy ( Protein from serial urine collections taken over a period of 16-20 weeks (based on the last menstrual cycle) is extracted from a urine sample by mixing with 2 volumes (based on urine volume) of acetone. It was first precipitated and kept at 4°C. Collect the precipitated protein and save the original sample in PBS. Resuspend using 1/20 of the volume. The sex of the carried fetus can be determined by amniocentesis or After birth, it was determined either by visual inspection.

The United Stake Government is a public health service Contract N0I-CP-41009, Public Health Services - Has rights to this invention pursuant to Grant CA38160 and CA25803.

The above is intended to illustrate the invention, and not to limit it. . Numerous changes and modifications may be made without departing from the true spirit and novel concept of the invention. can be done.

ABCD A [xlOl ABCDEF GHIJKL ABCDE FGHIJ KL M PJO Armor, Otsu ABCDEFGH FIG, 8゜ ABCD E F ABCD 7-ros97-118 Cell extract supernatant ABCD EFGH hσ/3° F/G, /4゜ Sa4+　-”- σ/7 p55raS-tsuk-,- p2iras-,- B anti-fes Oncogene residue position polypeptide sequence - fos 927-938 1GRGNFGEVFSGSrC273-284LG QGCFGEVWMG2 557-568 I, GQGCFGE'S 310-321 LGQGCFGE4LGfms 61B-629LGTGAF GKVYEAerb B 13B-149LGTGAFGT Engineering YKGmht 9 1-102 EngineeringGSGSFGTVYGKraf 30-41 1GSGSFGT VYGKabl 36B-379LGGGQYGEVYEGmos 100-1 1. I LGSGGFGSVYKAEσ2θ Oncogene residue position polypeptide sequence F 0B-722FP IKWT APEAALYGRF/θ2/.

Conserved 1 kinase part 3 Refusal 1152-1167 LMQRCWEYDPH lie 5Fsrc 494-5 09 LMCQCWRKDPEERPTF Engineering 770-793 LMKLCWK KDPDE available Ffar 53L-546AMEQTWRLDPEERPTFf ms 910-933 FMQACWALEPTRRPTFerb B 366 -381 Engineering MVKCWMIDADSRPKFmht 316-331 LVA DCLKKVREERPLEraf 255-270 LVADCVKKVKE ERPTFabl 591-606 LMRACWQWNPSDRPSFmos 344-359 1 unit QSCWEARGLQRPTFrel 3B2-397 TLH5CWQQLYSPSPSAF/G, 22° FIG, 26° FIG, 2? FIG, 28° FIG, 29° ABCDEFGHIJ to L FIG, 30° ABCD E FG HIJ L FIG. 3/.

F’G, 32° ”　BTGF　ru　erbA FIG, 33° 2! ! BTGF ras erbA Fθ, 34° oB BTGF rag erbA m35゜ 0,! ! f3TGF ras erbAFIG, 38° RG, 39° D70 1YR FIG 40゜ ψoFF, ”>e%1~-~01~Tsutsururorororozu Tsutsutsu!~-〇Hetsushu-〇 〜〜唖−G〜〜−−〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜〜 Rororororo0000000000roQOro0roOrororororo0~O trick 0roro0 60 CI Cl Rorororo Ro ~ Roro ~ to 0 Roro ~ Φ ~ ~ ~ - Roki ~08°. 〜〜〜〜. . . 〜〜. 〜〜〜〜〜〜〜. ~. . 〜〜〜〜〜〜〜〜〜 ~. . . . . . . −. . 6. . . . -0-0°.

80°. oo-0-0°. . . . . NC%J. . . . -0°. . .

1st -〇　ψ pipe　　〇　rumor　-gu B 8°. . . . . . . . . . . . . .　Roro Sen 0. . . . . . ~αI;]Yo Rorororo OO Rorororo 0 Roroo Qo-～-Rorororo〇-RoroORoro RO 0 RO RO T.D. FIG, 43° 1 2 3456789+01112131415i6F/G 44σ.

F/G 44 power.

international search report

Claims (12)

[Claims] 1. A method of assaying a body sample for indicators of exposure to carcinogens, the method comprising: including below (a) contacting the body sample with a receptor molecule, wherein the receptor is ( i) a protein ligand and (ii) about 7 to about 40 amino acids corresponding to a portion of the protein. binds both to a peptide having an amino acid residue sequence containing a amino acid, and (b) detecting the amount of protein/receptor complex, if any, in the body sample; Determining the level of such a protein here has a high level compared to the level in a normal sample. The protein directs exposure of the body sample to carcinogens. 2. 2. The method of claim 1, wherein said body sample is selected from the group consisting of blood, tissue, and urine. 3. 2. The method of claim 1, wherein said assay is an immunoblot. 4. If the receptor molecule is a polyclonal antibody, oligoclonal antibody, or monochrome antibody, 2. The method of claim 1, wherein the antibody is selected from the group consisting of natural antibodies and fragments thereof. 5. The receptor molecule is associated with (a) H-ras oncogene-associated p21 and (b) There is a column] and [There is an array] From left to right and from the N-terminus to the C-terminus, as indicated by the formula chosen from 2. The method of claim 1, wherein the method binds to both polypeptides having an amino acid residue sequence. 6. A method for assaying for indicators of exposure to carcinogens in a body sample, the method comprising: (a) contacting the body sample with a receptor molecule, wherein the receptor is (i ) H-ras oncogene associated p21 or a nucleic acid sequence corresponding thereto; and (ii) [There is an array] and [There is an array] from left to right and from the N-terminus to the C-terminus, as indicated by the formula chosen from and a polypeptide having an amino acid residue sequence or a nucleic acid sequence corresponding thereto. (b) the amount of said body sample that bound, and (b) if necessary formed a complex with said receptor. determine the level of the protein or its corresponding nucleic acid sequence by detecting thing, wherein said protein or said nucleic acid sequence corresponding thereto compared to said level in a normal sample. High levels of are indicative of exposure to the carcinogen. including. 7. Indicators of cancer in the body sample prior to the manifestation of clinical signs of cancer in the individual from which the body sample is derived. (a) contacting the body sample with a receptor molecule. wherein said receptor has (i) a protein ligand and (ii) a portion of said protein. A peptide having an amino acid residue sequence containing about 7 to about 40 corresponding amino acids combined with both, and (b) if necessary, by detecting the amount of protein/receptor complexes in the body sample; determining the level of the protein; where the level of the protein is compared to that in a normal individual; High levels are an indication of cancer. including. 8. 8. The method of claim 7, wherein said body sample is selected from the group consisting of blood, tissue, and urine. 9. 8. The method of claim 7, wherein said assay is an immunoblot. 10. If the receptor molecule is a polyclonal antibody, an oligoclonal antibody, or a monoclonal antibody, 8. The method of claim 7, wherein the method is selected from the group consisting of local antibodies or fragments thereof. 11. The receptor molecules are (a) H-ras oncogene associated p21 and (b) There is an array] and [There is an array] from left to right and from the N-terminus to the C-terminus, as indicated by the formula selected from 8. The method of claim 7, wherein the method binds to both polypeptides having an amino acid residue sequence. 12. Indication of cancer in a body sample prior to the manifestation of clinical signs of cancer in the individual from which the body sample is derived. 1. A method of assaying for a substance comprising: (a) contacting the body sample with a receptor molecule; wherein said receptor is (i) H-ras oncogene associated p21 or its like; a nucleic acid sequence corresponding thereto; and (ii) [There is an array] and [There is an array] From left to right and from the N-terminus to the C-terminus, as indicated by the formula chosen from a polypeptide having an amino acid residue sequence or a nucleic acid sequence corresponding thereto; and (b) detecting the amount of the body sample component that has formed a complex with the receptor. determining the level of said protein or said nucleic acid sequence corresponding thereto; , higher levels of the protein or the corresponding nucleic acid sequence compared to normal samples are associated with the carcinogenic Indicates exposure to a substance. including.